Compositions and methods for treating blood disorders

Abstract

To provide new therapies for preventing, reducing the risk of developing, and treating blood disorders.SOLUTION: Provided is a method of preventing, reducing the risk of developing, or treating a hematological disorder comprising administering a C1q inhibitor to a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This patent application claims the benefit of U.S. Provisional Patent Application No. 62 / 916,492, filed October 17, 2019, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Blood disorders affect millions of people worldwide each year, regardless of age, race, gender, or socioeconomic status. Men, women, and children of all backgrounds live with complications associated with these conditions, many of which are potentially life-threatening. Blood disorders, commonly referred to as hematological disorders, are difficult to treat and represent a growing health concern in terms of both mortality and the cost of patient care. It is estimated that complications from deep vein thrombosis (DVT) kill more people each year than breast cancer, car accidents, and HIV combined.

[0003] Blood disorders can affect any of the three major components of blood: red blood cells, white blood cells, or platelets. Blood disorders can also affect the liquid portion of blood, known as plasma. Some blood disorders reduce the number of cells in the blood. For example, individuals with leukopenia have a reduced number of white blood cells and are more susceptible to infections. New therapies to treat blood disorders are needed.

[0004] Currently, there is no cure for blood disorders. The molecular mechanisms of blood cell homeostasis and the pathology of blood disorders are unknown. Therefore, new therapies are needed to prevent, reduce the risk of developing, and treat blood disorders. Summary of the Invention

[0005] The present disclosure generally relates to blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or The present invention relates to a method for preventing, reducing the risk of developing, or treating antiphospholipid syndrome (APS), an autoimmune disorder (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), an infectious disease (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus, e.g., SARS-CoV-2 (COVID)), an immune complex disease (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or a drug-induced hematological disorder (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin), comprising administering to a subject an inhibitor of the complement pathway.

[0006] Blood disorders may be referred to as hematological disorders. Hematological disorders have diverse etiologies, but some may be caused by mutations and / or autoantibodies that inactivate complement regulatory proteins and mutations that directly activate the complement cascade. For example, complement mutations typically cause uninhibited complement activation, occurring in platelets, neutrophils, monocytes, and their aggregates, as well as red blood cells and endothelial cells. Complement activation in these cells results in the release of cell-derived microvesicles that may express complement and tissue factor, thereby promoting inflammation. Complement deposition on red blood cells causes hemolysis and the release of prothrombotic red blood cell-derived microvesicles. Complement deposition can also occur on cells within the vascular system, such as endothelial cells, or in highly vascularized tissues, such as capillary beds, glomeruli, and alveoli, which can lead to vascular damage in many organs. Complement activation can be prevented by inhibitors that block activation of the complement cascade. Such inhibitors may block the expression of specific complement proteins in blood cells or in associated cells and vascular tissues, interfere with signaling molecules that induce complement activation, upregulate the expression of complement inhibitors in blood cells or in associated cells and vascular tissues, or otherwise interfere with the role of complement in blood or hematological disorders.

[0007] Therefore, inhibition of the complement activation pathway can be used to treat blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP)). , thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronaviruses, e.g., SARS-CoV-2 (COVID)), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin). Specifically, anti-C1q, anti-C1r, and anti-C1s antibodies can prevent autoantibodies from triggering complement activation.

[0008] The present disclosure generally relates to the treatment of blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia, and thrombosis) by inhibiting classical complement activation, for example, by inhibiting complement factors Clq, Clr, or Cls, for example, through administration of antibodies, such as monoclonal, chimeric, humanized, human antibodies, antibody fragments, antibody derivatives, that bind to one or more of these complement factors. The present invention relates to methods for preventing, reducing the risk of developing, or treating HIV-1 thrombocytopenic purpura (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronaviruses, e.g., SARS-CoV-2 (COVID)), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin). In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment, such as a Fab fragment.

[0009] In some embodiments, the activity of complement factors such as C1q, C1r, or C1s is inhibited to prevent activation of the classical complement pathway and to treat hematologic disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP)). , immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronaviruses, e.g., SARS-CoV-2 (COVID)), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin). Inhibition of the classical complement pathway leaves the lectin and alternative complement pathways intact to carry out their normal immune functions.Blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and Disclosed herein are methods related to neutralizing complement factors such as C1q, C1r, or C1s in antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronaviruses, e.g., SARS-CoV-2 (COVID)), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin).

[0010] In certain aspects, the term "blood disorder" as used herein refers to a blood disorder (e.g., cold agglutinin hemolytic anemia (cold agglutinin hemolytic anemia (cold agglutinin disease)), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, thrombosis, vasculitis, thrombocytopenia, thrombosis, vasculitis, lupus nephritis, thrombosis ... Disclosed are methods for preventing, reducing the risk of developing, or treating glomerulonephritis and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronaviruses such as SARS-CoV-2 (COVID)), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin) comprising administering to a subject an inhibitor of the complement pathway.

[0011] Disclosed herein is a method for inhibiting complement activation in blood disorders, comprising administering an antibody, such as an anti-Clq antibody, an anti-Clr antibody, or an anti-Cls antibody, to a patient suffering from harmful complement activation. The method may further comprise administering a therapeutic agent. In certain preferred embodiments, the antibody binds to C1q, C1r, or C1s and inhibits complement activation.

[0012] In some aspects, methods for preventing, reducing the risk of developing, or treating a blood disorder are disclosed. Such methods include administering a C1q inhibitor to a subject. Numerous embodiments are further provided that may be applied to any aspect of the invention described herein. For example, in some embodiments, the C1q inhibitor is an antibody, an aptamer, an antisense nucleic acid, or a gene editing agent. In some embodiments, the inhibitor is an anti-C1q antibody. The anti-C1q antibody may inhibit the interaction between C1q and autoantibodies, or between C1q and C1r, or between C1q and C1s, or may promote the clearance of C1q from the circulation or tissues. In some embodiments, the anti-C1q antibody has a dissociation constant (K) in the range of 100 nM to 0.005 nM or less than 0.005 nM. DIn some embodiments, the anti-C1q antibody binds to C1q with a binding stoichiometry ranging from 20:1 to 1.0:1 or less than 1.0:1, a binding stoichiometry ranging from 6:1 to 1.0:1 or less than 1.0:1, or a binding stoichiometry ranging from 2.5:1 to 1.0:1 or less than 1.0:1. The antibody specifically binds to C1q and exhibits its biological activity, for example, (1) C1q binding to autoantibodies, (2) C1q binding to C1r, (3) C1q binding to C1s, (4) C1q binding to IgM, (5) C1q binding to phosphatidylserine, (6) C1q binding to pentraxin-3, (7) C1q binding to C-reactive protein (CRP), (8) C1q binding to globular C1q receptor (gC1qR), (9) C1q binding to complement receptor 1 (CR1). q binding, (10) C1q binding to beta-amyloid, (11) C1q binding to calreticulin, (12) C1q binding to apoptotic cells, or (13) C1q binding to B cells, or (1) activation of the classical complement pathway, (2) decreased lysis and / or decreased C3 deposition, (3) activation of antibody and complement-dependent cytotoxicity, (4) CH50 hemolysis, (5) decreased erythrocyte lysis, (6) decreased erythrophagocytosis, (7) decreased dendritic cell infiltration, (8) complement-mediated erythrocyte lysis (9) inhibition of lymphocyte infiltration, (10) reduction in macrophage infiltration, (11) reduction in antibody deposition, (12) reduction in neutrophil infiltration, (13) reduction in platelet phagocytosis, (14) reduction in platelet lysis, (15) improvement in graft survival, (16) reduction in macrophage-mediated phagocytosis, (17) reduction in autoantibody-mediated complement activation, (18) reduction in red blood cell destruction due to transfusion reactions, (19) reduction in alloantibody-mediated red blood cell lysis, (20) reduction in hemolysis due to transfusion reactions, (21) reduction in alloantibody-mediated platelet lysis (22) improvement of anemia, (23) reduction in eosinophilia, (24) reduction in C3 deposition on red blood cells (e.g., reduction in C3b, iC3b, etc. deposition on RBCs), (25) reduction in C3 deposition on platelets (e.g., reduction in C3b, iC3b, etc. deposition on platelets), (26) reduction in anaphylatoxin production, (27) reduction in autoantibody-mediated rash formation, (28) reduction in autoantibody-induced lupus erythematosus, (29) reduction in red blood cell destruction due to transfusion reactions, (30) reduction in platelet lysis due to transfusion reactions.The antibody may neutralize (31) reduced mast cell activation, (32) reduced mast cell histamine release, (33) reduced vascular permeability, (34) reduced complement deposition on graft endothelium, (35) B cell antibody production, (36) dendritic cell maturation, (37) T cell proliferation, (38) cytokine production, (39) microglial activation, (40) the Arthus reaction, (41) reduced anaphylatoxin production in graft endothelium, or (42) activation of complement receptor 3 (CR3 / C3)-expressing cells. In some embodiments, CH50 hemolysis includes human CH50 hemolysis. The antibody may be capable of neutralizing at least about 50% to about 100% of human CH50 hemolysis. The antibody may be capable of neutralizing about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of human CH50 hemolysis. The antibody may be capable of neutralizing at least 50% of CH50 hemolysis at a dose of less than 150 ng / ml, less than 100 ng / ml, less than 50 ng / ml, or less than 20 ng / ml.

[0013] In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a human antibody, a chimeric antibody, a monovalent antibody, a multispecific antibody, or an antibody fragment, or an antibody derivative thereof. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment, such as a Fab fragment. Examples of antibody fragments are Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, and single-chain antibody molecules. In some embodiments, the antibody comprises a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO:5, HVR-L2 having the amino acid sequence of SEQ ID NO:6, and HVR-L3 having the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody comprises a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO:9, HVR-H2 having the amino acid sequence of SEQ ID NO:10, and HVR-H3 having the amino acid sequence of SEQ ID NO:11. In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% identity to an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, wherein the light chain variable domain comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the light chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 4 and 35-38. In some embodiments, the antibody comprises a heavy chain variable domain comprising an amino acid sequence having at least about 95% identity to an amino acid sequence selected from SEQ ID NOs: 8 and 31-34, wherein the heavy chain variable domain comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 8 and 31-34. In some embodiments, the antibody is an antibody fragment comprising a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40. The antibody may be administered by parenteral injection or infusion, such as subcutaneous or intramuscular injection, or intravenous injection or infusion.

[0014] In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of between 10 mg / kg and 150 mg / kg. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of between 10 mg / kg and 20 mg / kg, 20 mg / kg and 30 mg / kg, 30 mg / kg and 40 mg / kg, 40 mg / kg and 50 mg / kg, 50 mg / kg and 60 mg / kg, 60 mg / kg and 70 mg / kg, 70 mg / kg and 80 mg / kg, 80 mg / kg and 90 mg / kg, 90 mg / kg and 100 mg / kg, 100 mg / kg and 110 mg / kg, 110 mg / kg and 120 mg / kg, 120 mg / kg and 130 mg / kg, 130 mg / kg and 140 mg / kg, or 140 mg / kg and 150 mg / kg. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose between 75 mg / kg and 100 mg / kg. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 10 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 75 mg / kg, 80 mg / kg, 90 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg, 130 mg / kg, 140 mg / kg, or 150 mg / kg. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 75 mg / kg. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 100 mg / kg. The antibody may be administered weekly, biweekly, or monthly. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 75 mg / kg. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 100 mg / kg. The antibody can be administered once a week, once every two weeks, once every three weeks, or once a month. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 75 mg / kg once a week. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 75 mg / kg once every two weeks.In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 75 mg / kg once every three weeks. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 75 mg / kg once a month. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 100 mg / kg once a week. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 100 mg / kg every two weeks. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 100 mg / kg once every three weeks. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 100 mg / kg once a month. In some embodiments, the antibody is administered to the subject by subcutaneous or intramuscular injection at a dose between 1 mg / kg and 10 mg / kg. In some embodiments, the antibody is administered to a subject by subcutaneous or intramuscular injection at a dose of between 1 mg / kg and 3 mg / kg, 3 mg / kg and 5 mg / kg, 5 mg / kg and 7 mg / kg, or 7 mg / kg and 10 mg / kg. In some embodiments, the antibody is administered daily, every other day, weekly, every two weeks, every three weeks, or monthly.

[0015] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is administered to a subject by intravenous injection or infusion, intramuscular injection, or subcutaneous injection. In some embodiments, the antibody fragment is administered at a dose between 0.1 mg / kg and 50 mg / kg. In some embodiments, the antibody fragment is administered at a dose between 0.1 mg / kg and 1 mg / kg, 1 mg / kg and 5 mg / kg, 5 mg / kg and 10 mg / kg, 10 mg / kg and 15 mg / kg, 15 mg / kg and 20 mg / kg, 20 mg / kg and 25 mg / kg, 25 mg / kg and 30 mg / kg, 30 mg / kg and 35 mg / kg, 35 mg / kg and 40 mg / kg, 40 mg / kg and 45 mg / kg, or 45 mg / kg and 50 mg / kg. In some embodiments, the antibody fragment is administered at a dose between 0.3 mg / kg and 10 mg / kg. In some embodiments, the antibody fragment is administered daily, every other day, weekly, biweekly, or monthly. In some embodiments, the antibody fragment is administered at an initial, pre-dosing dose that is higher than the daily, biweekly, weekly, biweekly, or monthly dose. In some embodiments, the initial, pre-dosing dose is between 3 mg / kg and 50 mg / kg. In some embodiments, the initial, pre-dosing dose is between 3 mg / kg and 5 mg / kg, 5 mg / kg and 10 mg / kg, 10 mg / kg and 15 mg / kg, 15 mg / kg and 20 mg / kg, 20 mg / kg and 25 mg / kg, 25 mg / kg and 30 mg / kg, 30 mg / kg and 35 mg / kg, 35 mg / kg and 40 mg / kg, 40 mg / kg and 45 mg / kg, or 45 mg / kg and 50 mg / kg. In some embodiments, the initial, pre-dosing dose is between 3 mg / kg and 20 mg / kg. In some embodiments, the antibody fragment has a shorter half-life compared to its corresponding full-length antibody, e.g., the antibody fragment is rapidly cleared, thereby sparing C1q activity outside the blood space of the subject, or the antibody selectively inhibits C1q within the blood space of the subject, thereby sparing C1q activity outside the blood space of the subject. In some embodiments, the blood space is limited within a blood vessel, such as an artery, arteriole, capillary, venule, or vein. The blood space may contain serum, platelets, endothelial cells, blood cells, or hematopoietic cells.In some embodiments, inhibiting C1q in the blood space of a subject reduces tissue damage in highly vascularized tissues. Examples of highly vascularized tissues are the kidney, alveoli, capillary beds, or glomeruli.

[0016] In some embodiments, the blood disorder is a complement-mediated blood disorder. In some embodiments, the blood disorder is cold agglutinin hemolytic anemia (cold agglutinin disease), cold antibody hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty's disease. The infection may be caused by a thrombocytopenic syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, systemic lupus erythematosus (SLE), glomerulonephritis, antiphospholipid syndrome (APS), an infectious disease, or a drug-induced hematological disorder. The infectious disease may be pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), or a coronavirus. Examples of coronaviruses are selected from SARS-CoV, MERS-CoV, HCoV, HKU1, and SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the subject has a SARS-CoV-2 infection confirmed by reverse transcription-polymerase chain reaction (RT-PCR) from a respiratory tract or blood sample. The blood disorder can be cold agglutinin hemolytic anemia (cold agglutinin disease), warm autoimmune hemolytic anemia (WAIHA), lupus nephritis, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), or immune thrombocytopenic purpura (ITP). Examples of drug-induced hematological disorders are aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, and thrombocytopenia.

[0017] In some aspects, methods for preventing, reducing the risk of developing, or treating a hematological disorder are disclosed. Such methods include administering an inhibitor of the classical complement pathway to a subject, the subject comprising a blood cavity, wherein the inhibitor selectively inhibits the classical complement pathway within the blood cavity of the subject, thereby sparing complement activity within the tissue. Numerous embodiments are further provided that may be applied to any aspect of the invention described herein. [Brief explanation of the drawings]

[0018] [Figure 1A] We demonstrate that anti-C1q antibody (Mab1) effectively halts processes associated with both intravascular and extravascular RBC lysis in CAD. We demonstrate that anti-C1q antibody inhibits C1q, C4d, and C3b binding / activation on the RBC surface in the presence of serum from patients with CAD, preventing extravascular lysis. [Figure 1B] We demonstrate that anti-C1q antibodies (Mab1) effectively halt processes associated with both intravascular and extravascular RBC lysis in CAD. We demonstrate that anti-C1q antibodies block C5-C9-mediated lysis of red blood cells initiated by serum from CAD patients, preventing intravascular lysis. [Figure 2A] Figures 2A and 2B show that anti-C1q (e.g., Mab1) and anti-C1s (e.g., TNT009) antibodies inhibit complement-mediated hemolysis. Figure 2A shows that both anti-C1q and TNT009 inhibit antibody / complement-induced lysis of red blood cells. Figure 2B shows that only anti-C1q antibodies inhibit upstream binding of C1q to target cells. C1q binding to RBCs is unaffected by TNT009. C1q is one of the three major opsonin / immune cell ligands deposited on red blood cells. [Figure 2B]Figures 2A and 2B show that anti-C1q (e.g., Mab1) and anti-C1s (e.g., TNT009) antibodies inhibit complement-mediated hemolysis. Figure 2A shows that both anti-C1q and TNT009 inhibit antibody / complement-induced lysis of red blood cells. Figure 2B shows that only anti-C1q antibodies inhibit upstream binding of C1q to target cells. C1q binding to RBCs is unaffected by TNT009. C1q is one of the three major opsonin / immune cell ligands deposited on red blood cells. [Figure 3] Anti-C1q antibodies (e.g., Mab1) have been shown to selectively inhibit the classical complement cascade and, unlike anti-C5, leave the lectin and alternative pathways intact, allowing normal immune function. [Figure 4] Figure 1 shows serum biomarkers of complement depletion / consumption in CAD patients. Decreases in C4 and C2, but not C5, indicate hyperactivation of the early complement cascade along with consumption of early complement components. [Figure 5] 1 shows that subcutaneous administration of anti-C1q antibody fragments (eg, FabA) inhibits RBC lysis in primates. [Figure 6A] 1 shows dose-dependent inhibition of serum hemolysis and complement deposition with anti-C1q antibody (Mab2) and FabA in samples from CAD patients. The effect of Mab2 is shown. [Figure 6B] 1 shows dose-dependent inhibition of serum hemolysis and complement deposition with anti-C1q antibody (Mab2) and FabA in samples from CAD patients. Effect of FabA is shown. [Figure 7A]This shows that PF4 / heparin activates complement via the classical pathway. This graph shows complement activation under different incubation conditions. Plasma from healthy donors was incubated with EDTA (10 mM) or EGTA (10 mM) ± MgCl2 (10 mM) or with buffer, followed by incubation with PF4 / heparin, and complement activation was measured by an antigen-C3e capture ELISA assay. ***p<0.0001. Results are shown from a representative experiment involving three donors tested on three different occasions. [Figure 7B] This shows that PF4 / heparin activates complement via the classical pathway. This is a graph showing complement activation under different incubation conditions. Plasma from healthy donors was incubated with or without Cl- inhibitors (10 and 20 IU / mL) and then incubated with PF4 / heparin, and complement activation by PF4 / heparin was determined by antigen-C3c capture ELISA assay. ***p<0.0001. [Figure 7C] This shows that PF4 / heparin activates complement via the classical pathway. Figure 1 shows histograms showing binding of anti-PF4 / heparin (KKO) to B cells under various incubation conditions. Overlapping peaks represent the buffer control (striped line), followed by PF4, PF4 / heparin + EDTA, PF4 / heparin + EGTA + MgCl2, and PF4 / heparin + EGTA. Peak 1 represents PF4 / heparin. [Figure 7D] Figure 1 shows that PF4 / heparin activates complement via the classical pathway. Figure 2 shows histograms showing binding of anti-C3e to B cells under various incubation conditions. Overlapping peaks represent PF4 / heparin + EDTA, PF4 / heparin + EGTA, PF4 / heparin + EGTA + MgCl2, and buffer control (striped lines), and PF4. Peak 1 represents PF4 / heparin. [Figure 7E]This shows that PF4 / heparin activates complement via the classical pathway. This graph shows complement activation in the presence of various antibodies. Plasma from healthy donors was incubated with various concentrations of anti-Clq, anti-MBL, or control antibodies (0-100 μg / mL) before the addition of PF4 / heparin. Complement activation by PF4 / heparin was determined by an antigen-C3c capture ELISA assay. *p<0.05, **p<0.001, ***p<0.0001 compared to the condition where no antibody was added. Results are shown from a representative experiment involving three donors tested on three different occasions. [Figure 7F] Figure 1 shows that PF4 / heparin activates complement via the classical pathway. Figure 2 shows histograms showing binding of anti-PF4 / heparin to B cells under various incubation conditions. Peaks represent buffer control (striped line), anti-Clq+PF4 / heparin (peak 1), anti-MBL+PF4 / heparin (peak 2), PF4 / heparin (peak 3), and MS IgG1+PF4 / heparin (peak 4). [Figure 7G] Figure 1 shows that PF4 / heparin activates complement via the classical pathway. Figure 2 shows histograms showing binding of anti-C3c to B cells under various incubation conditions. Peaks represent buffer control (striped line), anti-Clq+PF4 / heparin (peak 1), anti-MBL+PF4 / heparin (peak 2), PF4 / heparin (peak 3), and MS IgG1+PF4 / heparin (peak 4). [Figure 8] This shows that complement activation by PF4 / heparin correlates with plasma / serum IgM levels. Figure 8 is a graph showing PF4 / heparin-induced C' activation (determined by an ELISA-based antigen capture assay) by different donors and their plasma IgM levels (quantified by proteomic analysis). For each point on the x-axis, the left bar represents C3e and the right bar represents IgM. [Figure 9] Serum free FabA levels in animals dosed at 5+1 mg / kg and 5+2 mg / kg are shown. Lower limit of quantitation = 5 ng / mL. [Figure 10] Figure 1 shows the reduction in free C1q in plasma from animals treated with 5 + 2 mg / kg FabA. Lower limit of quantitation = 1.1 μg / mL. [Figure 11] 1 shows that serum hemolysis was inhibited after repeated daily subcutaneous administration of FabA. [Figure 12A] Clearance data for Mab1 and FabA are shown, showing that Mab1 15mpk IV results in peak serum free Mab1 levels of 250,000 ng / mL. Free drug levels remain elevated through day 4 and decline to undetectable levels by day 5. [Figure 12B] Clearance data for Mab1 and FabA are shown. FabA 10 mpk IV yields peak drug levels of 12,000 ng / mL, which are cleared very rapidly with drug levels dropping below detection limits by 8 hours. The estimated half-life of the Fab molecule is 2-3 hours. [Figure 12C] Clearance data for Mab1 and FabA are shown, showing that FabA 3mpk SC exhibits a very modest increase in free drug levels, measurable at 24 hours after a single dose. [Figure 13] Figure 1 shows complement deposition in samples from AIHA patients and inhibition of deposition with anti-C1q antibody (Mab2). DETAILED DESCRIPTION OF THE INVENTION

[0019] Overview The present disclosure generally relates to blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis). and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronaviruses, e.g., SARS-CoV-2 (COVID)), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia resulting from drugs such as penicillin, quinine, or heparin), the method comprising administering to a subject an inhibitor of the complement pathway.

[0020] The blood disorders of the present invention have a variety of etiologies; however, they are typically characterized by uninhibited complement activation on blood components and cells, as well as on related cells within the vasculature and highly vascularized tissues. Complement activation on these cells results in the deposition of complement components, which can lead to immune cell recruitment and attack. It also results in the release of cell-derived microvesicles that can express complement and tissue factor, thereby promoting inflammation. Complement deposition on red blood cells can cause intravascular or extravascular hemolysis and / or the release of prothrombotic red blood cell-derived microvesicles. Complement deposition can also occur on cells within the vasculature, such as endothelial cells, or within highly vascularized tissues, such as capillary beds, glomeruli, and alveoli, which can lead to vascular damage in many organs. Complement deposition on red blood cells can also result in enhanced extravascular clearance. Complement activation can be prevented by inhibitors that block activation of the complement cascade. Such inhibitors may block the expression of specific complement proteins in blood cells or in associated cells of the vasculature and highly vascularized tissues, interfere with signaling molecules that induce complement activation, upregulate the expression of complement inhibitors in blood cells or in associated cells and vascularized tissues, or otherwise interfere with the role of complement in blood or hematological disorders.

[0021] For example, patients with chronic hemolytic diseases often develop severe anemia. In cold agglutinin disease (CAD) and warm autoimmune hemolytic anemia (wAIHA), autoreactive antibodies against red blood cells (RBCs) trigger C1q binding and classical complement activation. Complement activation leads to RBC clearance, resulting in chronic anemia. Complement-mediated red blood cell damage occurs when C1q recognizes autoantibodies bound to RBCs, triggering the classical pathway to coat the RBCs with activated complement components (C1q, C4b, and C3b), and the complement-coated RBCs are removed from the circulation, resulting in anemia. In CAD and wAIHA, RBCs are coated with three major classical complement "opsonins," C1q, C4b, and C3b, which promote RBC clearance via "extravascular lysis." C1q, C4b, and C3b are recognized in the spleen and liver by the reticuloendothelial system for RBC removal. Furthermore, in CAD and wAIHA, RBCs are coated with C5b, which initiates membrane attack complex (MAC)-mediated lysis of red blood cells, resulting in direct intravascular RBC lysis. Anti-C1q effectively halts both the intravascular and extravascular processes involved in RBC lysis in CAD (Figures 1A-1B). Anti-C1q antibodies can inhibit the deposition of key "opsonins" / immune cell ligands (C1q, C4b, and C3b) of the complement cascade. Both anti-C1q antibodies (e.g., Mab1 antibody, comprising the heavy chain variable domain of SEQ ID NO: 3 and the light chain variable domain of SEQ ID NO: 7) and anti-C1s (e.g., TNT009) antibodies inhibit direct complement-mediated hemolysis (consistent with the inhibition of intravascular lysis) (Figure 2A), whereas only anti-C1q antibodies inhibit the upstream binding of C1q to target cells, the opsonin responsible for extravascular lysis (Figure 2B). Anti-C1s antibodies do not block C1q binding, anti-C3 will not block C1q or C4b binding to RBCs, and anti-C5 will not inhibit C1q, C4b, or C3b binding to RBCs. Only anti-C1q inhibits coating of RBCs by all three opsonins involved in extravascular hemolysis.

[0022] Inhibiting the complement pathway (for example, by anti-C1q antibody) stops complement deposition on cells in the vascular system or highly vascularized tissues. In blood disorders, C1q binds to components exposed to or by damaged tissues, causing complement activation, along with C1q, C4b, and C3b deposition on cell surfaces and further damage. By preventing C1q from binding to cells in blood cavities or highly vascularized tissues, it prevents further complement-mediated damage to tissues or organs. For example, lupus nephritis can be treated by preventing C1q activation on the surface of cells in the highly vascularized part of the kidney where blood filtration occurs.

[0023] Anti-C1q antibodies (e.g., Mab1 antibody, comprising a heavy chain variable domain of SEQ ID NO: 3 and a light chain variable domain of SEQ ID NO: 7) selectively inhibit the classical pathway, sparing normal immune function of the lectin and alternative pathways (Figure 3). In contrast, anti-C5, like anti-C3, inhibits the hemolytic activity of all three pathways (Figure 3). Unlike anti-C3 and anti-C5 antibodies, anti-C1q antibodies leave the lectin and alternative pathways intact, allowing normal immune function. Serum biomarkers of complement depletion / consumption in CAD patients provide additional assessment. Decreases in C4 and C2, but not C5, are consistent with chronic overactivation of the early complement cascade, along with consumption of early complement components (Figure 4). CAD can be treated by subcutaneous administration of anti-C1q antibodies (e.g., FabA, an anti-C1q Fab comprising a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40) to inhibit RBC lysis in primates (Figure 5).

[0024] The difference between administering a Fab versus a whole antibody to C1q is the degree of systemic C1q inhibition. Given the long half-life of a full-length antibody, the antibody remains in the blood space for a long time (e.g., several days) after administration of the full-length antibody. This allows the antibody to penetrate tissues and inhibit C1q activity throughout the body. For example, 10 mg / kg of a full-length antibody will persist in the blood space for several days, allowing time for tissue penetration and systemic C1q inhibition. In some situations, it may be preferable to limit C1q inhibition to the vascular compartment for the treatment of vascular disease (essentially a "local treatment" for the disease) while allowing C1q function elsewhere. For this purpose, Fab fragments with high affinity and a shorter half-life are administered subcutaneously or intravenously. For example, when 10 mg / kg (or 0.3 mg / Kg to 20 mg / Kg) of Fab is given IV, the free drug is cleared rapidly (≤8 hours) - however, the drug bound to C1q in the circulation persists, so that C1q remains inhibited for approximately 24 hours before it is replaced.

[0025] In one such application, CAD is a chronic, but usually non-life-threatening, disease that primarily occurs in elderly individuals. In such cases, there may be a safety benefit of selectively inhibiting C1q in the vascular space to protect RBCs while allowing C1q to exert its normal immune function elsewhere in the body. This goal can be achieved by subcutaneous self-administration of an anti-C1q monovalent Fab (e.g., an anti-C1q antibody Fab fragment ("FabA") comprising a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40). Due to the extremely high affinity of the monovalent Fab (10 pM), the drug remains tightly bound to C1q while C1q moves through the circulation. Free drug (unbound to C1q) rapidly clears from the circulation and does not enter tissues. The function of circulating C1q is restored with blood C1q turnover (24-48 hours) (Figure 5). The anti-C1q monovalent Fab can be administered subcutaneously, for example, daily. Anti-C1q monovalent Fab, administered subcutaneously at 0.3-10 mg / kg every 24 hours (or every other day, week, biweekly, or monthly, depending on how quickly the Fab construct is absorbed through the skin), can sufficiently inhibit complement activation on the surface of circulating RBCs, thereby preventing both intravascular and extravascular RBC lysis (in CAD, "extravascular" lysis occurs in the liver through Kupffer cell capture of complement-coated circulating RBCs). However, after administration, anti-C1q monovalent Fab (e.g., an anti-C1q antibody Fab fragment comprising a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40) selectively inhibits C1q in the blood cavity, thereby preventing complement deposition on circulating RBCs, while allowing tissue C1q to maintain normal immune function.

[0026] A Fab fragment of a high-affinity antibody to C1q with a short circulating half-life may be sufficient to suppress C1q activity in the blood space for 24 hours upon daily subcutaneous administration, which would limit the extent of systemic inhibition (i.e., inhibition of C1q in tissues) and thereby preserve the function of C1q outside the blood space.

[0027] Neutralization of the activity of complement factors such as C1q, C1r, or C1s inhibits classical complement activity and prevents complement-mediated disorders of the vascular compartment (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO-incompatible acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO-incompatible acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune hemolytic anemia). It delays or prevents thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronaviruses, e.g., SARS-CoV-2 (COVID)), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin). Inhibition of the classical complement pathway leaves the lectin and alternative complement pathways intact to perform their normal immune functions.Blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin hemolytic anemia (cold agglutinin disease)), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vascular Disclosed herein are methods related to neutralizing complement factors such as C1q, C1r, or C1s in inflammatory bowel disease, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin).

[0028] All sequences recited in this disclosure are incorporated by reference from U.S. Patent Application No. 14 / 933,517, U.S. Patent Application No. 14 / 890,811, U.S. Patent No. 8,877,197, U.S. Patent No. 9,708,394, U.S. Patent Application No. 15 / 360,549, U.S. Patent No. 9,562,106, U.S. Patent No. 10,450,382, U.S. Patent No. 10,457,745, International Patent Application No. PCT / US2018 / 022462 (each of which is incorporated by reference herein for the antibodies and related compositions it discloses).

[0029] In certain aspects, the term "blood disorder" as used herein refers to a blood disorder (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, loop thrombocytopenia, vasculitis, thrombocytopenic purpura, thrombocytopenia, thrombosis, vasculitis ... Disclosed are methods for preventing, reducing the risk of developing, or treating leukemia, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin), comprising administering to a subject an inhibitor of the complement pathway.

[0030] Full-length antibodies can be prepared using recombinant DNA engineering techniques. Such engineered versions include, for example, those created from natural antibody variable regions by insertions, deletions, or changes within or to the amino acid sequence of the natural antibody. Particular examples of this type include those engineered variable region domains containing at least one CDR from one antibody and, optionally, one or more framework amino acids and the remainder of the variable region domain from a second antibody. DNA encoding an antibody can be prepared by deleting all but the desired portion of DNA encoding a full-length antibody. DNA encoding a chimerized antibody can be prepared by recombining DNA encoding substantially or exclusively human constant regions with DNA encoding variable regions substantially or exclusively derived from variable region sequences of a mammal other than a human. DNA encoding a humanized antibody can be prepared by recombining DNA encoding variable regions other than the constant regions and complementarity-determining regions (CDRs) substantially or exclusively derived from corresponding human antibody regions with DNA encoding CDRs substantially or exclusively derived from a mammal other than a human.

[0031] Suitable sources of DNA molecules encoding antibodies include cells, such as hybridomas, that express full-length antibodies. For example, antibodies can be isolated from host cells that express expression vectors encoding the heavy and / or light chains of the antibody.

[0032] Antibody fragments and / or antibody derivatives can also be prepared by the use of recombinant DNA engineering techniques, including the manipulation and re-expression of DNA encoding antibody variable and constant regions. Standard molecular biology techniques can be used to modify, add, or delete additional amino acids or domains, as desired. Any changes to the variable or constant regions will still be encompassed by the terms "variable" and "constant" regions as used herein. In some instances, C H C so that translation of the 1 domain stops at the interchain cysteine. HPCR is used to generate antibody fragments by introducing a stop codon immediately after the codon encoding the interchain cysteine ​​of antibody C. Methods for designing suitable PCR primers are well known in the art and are described in detail in the literature for antibody C. H The sequence of one domain is readily available. In some embodiments, a stop codon can be introduced using site-directed mutagenesis techniques.

[0033] Antibodies of the present disclosure may be derived from any antibody isotype ("class"), including, for example, IgG, IgM, IgA, IgD, and IgE, and subclasses thereof (including, for example, IgG1, IgG2, IgG3, and IgG4). In certain preferred embodiments, the antibody heavy and light chains are derived from IgG. The antibody heavy and / or light chains may be derived from mouse IgG or human IgG. In certain other preferred embodiments, the antibody heavy and / or light chains are derived from human IgG1. In yet other preferred embodiments, the antibody heavy and / or light chains are derived from human IgG4.

[0034] In some embodiments, the inhibitor is an antibody such as an anti-Clq antibody, an anti-Clr antibody, or an anti-Cls antibody. An anti-C1q antibody can inhibit the interaction between C1q and autoantibodies, or between C1q and C1r, or between C1q and C1s. An anti-C1r antibody can inhibit the interaction between C1r and C1q, or between C1r and C1s. An anti-C1r antibody can inhibit the catalytic activity of C1r, or the processing of pro-C1r into active proteases. An anti-C1s antibody can inhibit the interaction between C1s and C1q, or between C1s and C1r, or between C1s and C2 or C4, or the anti-C1s antibody can inhibit the catalytic activity of C1s, or it can inhibit the processing of pro-C1s into active proteases. In some instances, anti-C1q, anti-C1r, or anti-C1s antibodies cause clearance of C1q, C1r, or C1s from the circulation or tissues.

[0035] The antibodies disclosed herein can be, for example, monoclonal antibodies that bind to mammalian C1q, C1r, or C1s, preferably human C1q, C1r, or C1s. The antibodies can be murine, human, humanized, chimeric, antibody fragments, or antibody derivatives thereof. In some embodiments, the antibodies are humanized. In some embodiments, the antibodies are antibody fragments, such as Fab fragments. The antibodies can be chimeric antibodies with sufficient human sequence suitable for administration to humans. The antibodies can be glycosylated or non-glycosylated; in some embodiments, the antibodies are glycosylated, e.g., with a glycosylation pattern generated by post-translational modification in CHO cells. In some embodiments, the antibodies are produced in E. coli.

[0036] The antibodies of the present disclosure may also be covalently linked to a therapeutic agent, for example, an anti-inflammatory protein, a neurotherapeutic agent, an antiviral agent, an antiparasitic agent, an antibacterial agent, an endocrine agent, a metabolic agent, a mitotoxin, a chemotherapeutic agent, or an siRNA.

[0037] In some embodiments, the anti-C1q, anti-C1r, or anti-C1s antibodies of the present disclosure reduce C3 deposition on red blood cells; for example, in some embodiments, the anti-C1q, anti-C1r, or anti-C1s antibodies of the present disclosure reduce C3b, iC3b, etc. deposition on RBCs. In some embodiments, the anti-C1q, anti-C1r, or anti-C1s antibodies of the present disclosure inhibit complement-mediated red blood cell lysis. The antibodies disclosed herein can reduce C3 deposition on platelets; for example, in some embodiments, the anti-C1q, anti-C1r, or anti-C1s antibodies of the present disclosure reduce C3b, iC3b, etc. deposition on platelets.

[0038] The antibodies of the present disclosure can bind to and inhibit the biological activity of C1q, C1r, or C1s, for example, (1) C1q binding to autoantibodies, (2) C1q binding to C1r, (3) C1q binding to C1s, (4) C1q binding to phosphatidylserine, (5) C1q binding to pentraxin-3, (6) C1q binding to C-reactive protein (CRP), (7) C1q binding to globular C1q receptor (gC1qR), (8) C1q binding to complement receptor 1 (CR1), (9) C1q binding to B-amyloid, or (10) C1q binding to calreticulin.In other embodiments, the biological activity of C1q is (1) activation of the classical complement pathway, (2) reduced lysis and / or reduced C3 deposition, (3) activation of antibody and complement dependent cytotoxicity, (4) CH50 hemolysis, (5) reduced erythrocyte lysis, (6) reduced erythrophagocytosis, (7) reduced dendritic cell infiltration, (8) inhibition of complement mediated erythrocyte lysis, (9) reduced lymphocyte infiltration, (10) reduced macrophage infiltration, (11) reduced antibody deposition, (12) Decreased neutrophil infiltration, (13) decreased platelet phagocytosis, (14) decreased platelet lysis, (15) improved graft survival, (16) decreased macrophage-mediated phagocytosis, (17) decreased autoantibody-mediated complement activation, (18) decreased red blood cell destruction due to transfusion reactions, (19) decreased alloantibody-mediated red blood cell lysis, (20) decreased hemolysis due to transfusion reactions, (21) decreased alloantibody-mediated platelet lysis, (22) improved anemia, (23) decreased eosinophilia, (24 ) Decreased C3 deposition on red blood cells (e.g., decreased C3b, iC3b, etc. deposition on RBCs), (25) Decreased C3 deposition on platelets (e.g., decreased C3b, iC3b, etc. deposition on platelets), (26) Decreased anaphylatoxin production, (27) Decreased autoantibody-mediated rash formation, (28) Decreased autoantibody-induced lupus erythematosus, (29) Decreased red blood cell destruction in transfusion reactions, (30) Decreased platelet lysis in transfusion reactions, (31) Obesity These include decreased cell activation, (32) decreased mast cell histamine release, (33) decreased vascular permeability, (34) decreased complement deposition on graft endothelium, (35) B cell antibody production, (36) dendritic cell maturation, (37) T cell proliferation, (38) cytokine production, (39) microglial activation, (40) Arthus reaction, (41) decreased anaphylatoxin production in graft endothelium, or (42) activation of complement receptor 3 (CR3 / C3)-expressing cells.

[0039] In some embodiments, the CH50 hemolysis includes human, mouse, and / or rat CH50 hemolysis. In some embodiments, the antibody is capable of neutralizing at least about 50% to at least about 95% of CH50 hemolysis. In some embodiments, the antibody is capable of neutralizing 50%, 60%, 70%, 80, 90%, or 100% of CH50 hemolysis. The antibody may also be capable of neutralizing at least 50% of CH50 hemolysis at a dose of less than 150 ng / ml, less than 100 ng / ml, less than 50 ng / ml, or less than 20 ng / ml.

[0040] Other in vitro assays for measuring complement activity include ELISA assays for measuring the cleavage products of complement components or complexes formed during complement activation. Complement activation via the classical pathway can be measured by monitoring serum C4d and C4 levels. Activation of the alternative pathway can be measured with ELISA by assessing the levels of Bb or C3bBbP complexes in the circulation. In vitro antibody-mediated complement activation assays can also be used to assess inhibition of C3a generation.

[0041] The antibodies of the present disclosure may be monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, human antibodies, chimeric antibodies, multispecific antibodies, antibody fragments thereof, or derivatives thereof. In some embodiments, the antibodies are humanized antibodies. In some embodiments, the antibodies are antibody fragments, such as Fab fragments.

[0042] An antibody of the disclosure can also be an antibody fragment, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, or a single-chain antibody molecule.

[0043] Disclosed herein are methods for administering a second agent, such as a second antibody or a second inhibitor, to a subject. The antibody can be an anti-Clq antibody, an anti-Clr antibody, or an anti-Cls antibody. The inhibitor can be an inhibitor of antibody-dependent cellular cytotoxicity, an inhibitor of the alternative complement activation pathway, and / or an inhibitor of the interaction between an autoantibody and an autoantigen.

[0044] In some embodiments, the therapeutic agent is selected from the group consisting of blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., , systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious disease (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex disease (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorder (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin)), comprising: (a) administering to the subject an antibody (i.e., anti-C1q, anti-C1r, or anti-C1s antibody), wherein the antibody is linked to a detectable label; (b) detecting the detectable label to measure the amount or location of C1q, C1r, or C1s in the subject;and (c) comparing the amount or location of one or more of C1q, C1r, or C1s with a reference, to identify a blood disorder (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), platelet Methods are provided in which the risk of developing thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin) is characterized based on comparison of the amount or location of one or more of C1q, C1r, or C1s to a reference. The detectable label may comprise a nucleic acid, an oligonucleotide, an enzyme, a radioisotope, biotin, or a fluorescent label. In some instances, antibodies can be labeled with a coenzyme such as biotin using the process of biotinylation. When biotin is used as a label, antibody detection is achieved by adding a protein such as avidin or its bacterial counterpart, streptavidin (either of which can be coupled to a detectable marker, such as the aforementioned dyes, fluorescent markers such as fluorescein, radioisotopes, or enzymes such as peroxidase). In some embodiments, the antibody is an antibody fragment (e.g., Fab, Fab'-SH, Fv, scFv, or F(ab')2 fragment) or an antibody derivative thereof;

[0045] The antibodies disclosed herein can also be linked to a labeling group, such as a radioisotope, a radionuclide, an enzyme group, a biotinyl group, a nucleic acid, an oligonucleotide, an enzyme, or a fluorescent label. The labeling group can be linked to the antibody via a spacer arm of any suitable length to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used to prepare such labeled antibodies.

[0046] Various routes of administration are contemplated. Such administration methods include, but are not limited to, topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intrathecal, intranasal, and intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Suitable antibodies include antibodies that bind to complement components C1q, C1r, or C1s. Such antibodies include monoclonal antibodies, human antibodies, chimeric antibodies, humanized antibodies, antibody fragments, and / or antibody derivatives thereof. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment, such as a Fab fragment.

[0047] In some embodiments, the antibody is a human monoclonal antibody that may be prepared, expressed, produced, or isolated by recombinant means, such as (a) an antibody isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) an antibody isolated from a host cell transformed to express the antibody, e.g., an antibody isolated from a transfectoma; (c) an antibody isolated from a recombinant combinatorial human antibody library; and (d) an antibody prepared, expressed, produced, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, if an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis), thus modifying the V and constant regions of the recombinant antibody. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0048] In some embodiments, the antibodies are humanized and / or chimeric monoclonal antibodies that can be established by immunizing rodents (e.g., mice, rats, hamsters, and guinea pigs) with (1) native complement components (e.g., C1q, C1r, or C1s) derived from enzymatic digestion of purified complement components from human plasma or serum, or (2) recombinant complement components expressed by either eukaryotic or prokaryotic systems, or derived fragments thereof. Other animals, such as non-human primates, transgenic mice expressing human immunoglobulins, and severe combined immunodeficient (SCID) mice engrafted with human B lymphocytes, can be used for immunization.

[0049] Polyclonal and monoclonal antibodies are naturally produced as immunoglobulin (Ig) molecules in the immune system's response to pathogens. The predominant form, the approximately 150 kDa IgG1 molecule, which has a concentration of 8 mg / ml in human serum, is composed of two identical approximately 50 kDa heavy chains and two identical approximately 25 kDa light chains.

[0050] Hybridomas can be generated by conventional procedures by fusing B lymphocytes from immunized animals with myeloma cells. Anti-C1q, C1r, or C1s antibodies can also be generated by screening recombinant single-chain Fv or Fab libraries from human B lymphocytes in a phage display system. The specificity of MAbs for human C1q, C1r, or C1s can be tested by enzyme-linked immunosorbent assay (ELISA), Western immunoblotting, or other immunochemical techniques.

[0051] The inhibitory activity of antibodies identified in the screening process against complement activation can be evaluated by hemolytic assay using naive rabbit or guinea pig RBCs for the alternative complement pathway, or sensitized chicken or sheep RBCs for the classical complement pathway. Hybridomas that exhibit specific inhibitory activity for the classical complement pathway are cloned by limiting dilution. Antibodies are purified for characterization of their specificity for human C1q, C1r, or C1s by the assays described above.

[0052] Based on the molecular structure of the variable region of an anti-C1q, C1r, or C1s antibody, molecular modeling and rational molecular design can be used to generate and screen small molecules that mimic the molecular structure of the antibody's binding region and inhibit the activity of C1q, C1r, or C1s. These small molecules can be peptides, peptidomimetics, oligonucleotides, or organic compounds. The mimetic molecules can be used as inhibitors of complement activation in inflammatory conditions and autoimmune diseases. Alternatively, large-scale screening procedures commonly used in the field can be used to isolate suitable small molecules from combinatorial compound libraries.

[0053] Suitable dosages can be determined by those skilled in the art using a variety of well-known methodologies, including the use of animal models and clinical trials, followed by conventional methodologies for determining optimal dosages, i.e., administering various dosages and determining which dose provides suitable efficacy without undesirable side effects.

[0054] Before the advent of recombinant DNA technology, proteolytic enzymes (proteases) that cleave polypeptide sequences were used to break down the structure of antibody molecules and determine which parts of the molecule are responsible for their various functions. Limited digestion with the protease papain cleaves antibody molecules into three fragments. Two of the fragments, known as Fab fragments, are identical and contain antigen-binding activity. The Fab fragments correspond to two identical arms of the antibody molecule, each of which contains the V-terminal end of the heavy chain. H and C H The other fragment, which does not contain antigen-binding activity, was initially observed to be readily crystallizable and for this reason was named the Fc fragment (fragment crystallizable).

[0055] Fab molecules contain the constant domain C H 2 and C H It is an artificial approximately 50 kDa fragment of an Ig molecule with a heavy chain lacking V3. L -V H and C L -C H1) Domain interactions underlie the structure of the two chains of the Fab molecule, which is C L and C H Fab and IgG are further stabilized by disulfide bridges between the V and V domains. Fab and IgG have six complementarity-determining regions (CDRs), three of which are V and V domains. L and V H The CDRs define the hypervariable antigen-binding site of an antibody. The highest sequence variation is found in LCDR3 and HCDR3, which in the natural immune system are V and V, respectively. L and J L Gene or V H , D H and J H They are generated by genetic rearrangement. LCDR3 and HCDR3 typically form the core of the antigen-binding site. The conserved regions connecting and presenting the six CDRs are called framework regions. In the three-dimensional structure of the variable domain, the framework regions form a sandwich of two opposing antiparallel β-sheets connected on the outside by the hypervariable CDR loops and on the inside by conserved disulfide bridges.

[0056] As used herein, blood disorders include, for example, cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, and the like. Disclosed are methods for protecting against or treating individuals suffering from inflammatory bowel disease, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin). Complement activation in blood and endothelial cells activates platelets, monocytes, neutrophils, red blood cells, and endothelial cells, which promotes thrombotic and inflammatory damage. These findings have broad relevance for various clinical conditions, especially blood disorders involving complement activation. Complement activation can be inhibited by contacting complement proteins with inhibitors or antagonists of the complement pathway. For example, the inhibitors can block the activation of the complement cascade, block the expression of specific complement proteins in blood cells, interfere with signaling molecules that induce complement activation, upregulate the expression of complement inhibitors in blood cells, and otherwise interfere with the role of complement in blood disorders. The ability to prevent complement activation has important relevance for maintaining normal blood function in various blood disorders.

[0057] The present disclosure also provides a method for detecting complement activation in an individual by: (a) administering to a subject an antibody from any of the embodiments, wherein the antibody is linked to a detectable label; (b) detecting the detectable label to measure the amount or location of the antibody in the subject; and (c) comparing the amount or location of the antibody with a reference, wherein the risk of developing a blood disorder associated with complement activation is characterized based on the comparison of the amount of antibody with the reference. For example, the detectable label can include a nucleic acid, an oligonucleotide, an enzyme, a radioisotope, biotin, or a fluorescent label (e.g., fluorescein, rhodamine, cyanine dye, or BODIPY). The detectable label can be detected using contrast agents for x-ray, CT, MRI, ultrasound, PET, and SPECT.

[0058] It should be understood that one, some, or all of the properties of the various embodiments described herein can be combined to form other embodiments of the compositions and methods provided herein. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein. These and other aspects of the compositions and methods provided herein will be apparent to those skilled in the art.

[0059] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0060] Anti-complement C1q antibodies The anti-C1q antibodies disclosed herein are potent inhibitors of C1q and can be dosed for continuous inhibition of C1q function for any period of time and then optionally discontinued to allow restoration of normal C1q function at a time when their activity may be important. The results obtained with the anti-C1q antibodies disclosed herein in animal studies can be readily translated to the clinic using humanized or human antibodies, and fragments and / or derivatives thereof.

[0061] C1q is a large 460 kDa multimeric protein consisting of 18 polypeptide chains (6 C1q A chains, 6 C1q B chains, and 6 C1q C chains). The C1r and C1s complement proteins bind to the C1q tail region to form the C1 complex (C1qr2s2).

[0062] The antibodies of the present disclosure specifically recognize complement factor C1q and / or C1q in the C1 complex of the classical complement activation pathway. The bound complement factor can be from any organism that has a complement system, including, but not limited to, any mammalian organism, such as a human, mouse, rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pig.

[0063] As used herein, a "C1 complex" refers to a protein complex that may include, but is not limited to, one C1q protein, two C1r proteins, and two C1s proteins (e.g., C1qr 2 s 2 ) refers to

[0064] The anti-C1q antibodies disclosed herein can inhibit C1 complex formation.

[0065] As used herein, "complement factor C1q" refers to both the wild-type sequence and the naturally occurring variant sequence.

[0066] A non-limiting example of a complement factor C1q recognized by the antibodies of the present disclosure is human C1q, which comprises three polypeptide chains A, B, and C:

[0067] C1q, chain A (homo sapiens), accession number Protein Database: NP_057075.1; GenBank number: NM_015991: >gi|7705753|ref|NP_057075.1|Complement C1q Subcomponent Subunit A Precursor [Homo sapiens] (SEQ ID NO: 1) MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAIRRN PPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYYFTFQVLSQWEICLSIVSSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA.

[0068] C1q, chain B (homo sapiens), accession number Protein Database: NP_000482.3; GenBank number: NM_000491.3: >gi|87298828|ref|NP_000482.3|Complement Clq Subcomponent Subunit B Precursor [Homo sapiens] (SEQ ID NO: 2) MMMKIPWGSIPVLMLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSAT RTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA.

[0069] C1q, chain C (homo sapiens), accession number Protein Database: NP_001107573.1; GenBank number: NM_001114101.1: >gi|166235903|ref|NP_001107573.1|Complement C1q Subcomponent Subunit C Precursor [Homo sapiens] (SEQ ID NO: 3) MDVGPSSLPHLGLKLLLLLLLLPLRGQANTGCYGIPGMPGLPGAPGKDGYDGLPGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPGMPGVPGPMGIPGEPGEEGRYKQKFQSVFT VTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD.

[0070] Thus, anti-C1q antibodies of the present disclosure may bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of the C1q protein. In some embodiments, anti-C1q antibodies of the present disclosure bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of human C1q or its homologues, e.g., mouse, rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pig C1q. In some embodiments, the anti-C1q antibody is a human antibody, a humanized antibody, a chimeric antibody, or a fragment or derivative thereof. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment, such as a Fab fragment.

[0071] Suitable antibodies include antibodies for use in blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (AP)). Included are antibodies that bind to complement C1q protein (i.e., anti-complement C1q antibodies, also referred to herein as anti-C1q antibodies and C1q antibodies), and nucleic acid molecules encoding such antibodies, for methods of preventing, reducing the risk of developing, or treating an autoimmune disorder (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), an infectious disease (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), an immune complex disease (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or a drug-induced hematological disorder (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin).

[0072] All sequences listed in the following 20 paragraphs are incorporated by reference from US Patent No. 9,708,394, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0073] Light and heavy chain variable domain sequences of antibody M1 (Mab2) The nucleic acid and amino acid sequences encoding the light chain variable and heavy chain variable domains of antibody M1 were determined using standard techniques. The amino acid sequence of the light chain variable domain of antibody M1 is:

[0074] [ka] is.

[0075] The hypervariable regions (HVRs) of the light chain variable domains are shown in bold and underlined text. In some embodiments, HVR-L1 of the M1 light chain variable domain has the sequence RASKSINKYLA (SEQ ID NO:5), HVR-L2 of the M1 light chain variable domain has the sequence SGSTLQS (SEQ ID NO:6), and HVR-L3 of the M1 light chain variable domain has the sequence QQHNEYPLT (SEQ ID NO:7).

[0076] The amino acid sequence of the heavy chain variable domain of antibody M1 is

[0077] [ka] is.

[0078] The hypervariable regions (HVRs) of the heavy chain variable domains are shown in bold and underlined text. In some embodiments, HVR-H1 of the M1 heavy chain variable domain has the sequence GYHFTSYWMH (SEQ ID NO:9), HVR-H2 of the M1 heavy chain variable domain has the sequence VIHPNSGSINYNEKFES (SEQ ID NO:10), and HVR-H3 of the M1 heavy chain variable domain has the sequence ERDSTEVLPMDY (SEQ ID NO:11).

[0079] The nucleic acid sequence encoding the light chain variable domain is GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAACAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAACATAATGAATACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO: 12) was determined to be

[0080] The nucleic acid sequence encoding the heavy chain variable domain is CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGTCTTCTGGCTACCATTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGTGATTCATCCTAATAGTGGTAGTATTAACTACAATGAGAAGTTCGAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCGGCGGTCTATTATTGTGCAGGAGAGAGAGATTCTACGGAGGTTCTCCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO: 13) was determined to be

[0081] Deposit of Substances The following materials have been deposited in accordance with the Budapest Treaty with the American Type Culture Collection, ATCC Patent Depository, 10801 University Blvd., Manassas, Va. 20110-2209, USA (ATCC):

[0082] [Table 1]

[0083] A hybridoma cell line (murine hybridoma C1qM1 7788-1(M)051613) producing the M1 antibody has been deposited with the ATCC under conditions ensuring that cultures will be available during the pendency of a patent application and for 30 years, or five years after the latest claim, or the life of the patent, whichever is longer. The deposit will be replaced if it becomes non-viable during that period. The deposit will be available as required by foreign patent laws in countries in which counterpart applications to this application, or their progeny, are filed. However, it should be understood that the availability of the deposit does not constitute a license to practice the invention in derogation of patent rights granted by government action.

[0084] Disclosed herein are methods for administering an anti-C1q antibody comprising a light chain variable domain and a heavy chain variable domain. The antibody can bind to at least human C1q, mouse C1q, or rat C1q. The antibody can be a humanized antibody, chimeric antibody, or human antibody. The antibody can be a monoclonal antibody, antibody fragment thereof, and / or antibody derivative thereof. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment, such as a Fab fragment. The light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3 of monoclonal antibody M1 produced by the hybridoma cell line deposited under ATCC accession number PTA-120399. The heavy chain variable domain comprises HVR-H1, HVR-H2, and HVR-H3 of monoclonal antibody M1 produced by the hybridoma cell line deposited under ATCC accession number PTA-120399.

[0085] In some embodiments, the amino acid sequences of the light chain variable domain and the heavy chain variable domain comprise one or more of SEQ ID NO: 5 for HVR-L1, SEQ ID NO: 6 for HVR-L2, SEQ ID NO: 7 for HVR-L3, SEQ ID NO: 9 for HVR-H1, SEQ ID NO: 10 for HVR-H2, and SEQ ID NO: 11 for HVR-H3.

[0086] The antibody may comprise a light chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO:4, preferably retaining HVR-L1 RASKSINKYLA (SEQ ID NO:5), HVR-L2 SGSTLQS (SEQ ID NO:6), and HVR-L3 QQHNEYPLT (SEQ ID NO:7). The antibody may comprise a heavy chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO:8, preferably retaining HVR-H1 GYHFTSYWMH (SEQ ID NO:9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO:10), and HVR-H3 ERDSTEVLPMDY (SEQ ID NO:11).

[0087] Disclosed herein is a method for administering an anti-C1q antibody that inhibits the interaction between C1q and autoantibodies. In a preferred embodiment, the anti-C1q antibody causes clearance of C1q from the circulation or tissues.

[0088] In some embodiments, an anti-C1q antibody of the present disclosure inhibits the interaction between C1q and C1s. In some embodiments, an anti-C1q antibody inhibits the interaction between C1q and C1r. In some embodiments, an anti-C1q antibody inhibits the interaction between C1q and C1s and between C1q and C1r. In some embodiments, an anti-C1q antibody inhibits the interaction between C1q and another antibody, e.g., an autoantibody. In preferred embodiments, an anti-C1q antibody causes clearance of C1q from the circulation or tissues. In some embodiments, an anti-C1q antibody inhibits each interaction with a stoichiometry of less than 2.5:1; 2.0:1; 1.5:1; or 1.0:1. In some embodiments, a C1q antibody inhibits an interaction, such as a C1q-C1s interaction, at approximately equimolar concentrations of C1q and anti-C1q antibody. In other embodiments, the anti-C1q antibody is less than 20:1; less than 19.5:1; less than 19:1; less than 18.5:1; less than 18:1; less than 17.5:1; less than 17:1; less than 16.5:1; less than 16:1; less than 15.5:1; less than 15:1; less than 14.5:1; less than 14:1; less than 13.5:1; less than 13:1; less than 12.5:1; less than 12:1; less than 11.5:1; less than 11:1

[0013] In certain embodiments, the anti-C1q antibody binds to C1q with a stoichiometry of less than 10:1; less than 10.5:1; less than 10:1; less than 9.5:1; less than 9:1; less than 8.5:1; less than 8:1; less than 7.5:1; less than 7:1; less than 6.5:1; less than 6:1; less than 5.5:1; less than 5:1; less than 4.5:1; less than 4:1; less than 3.5:1; less than 3:1; less than 2.5:1; less than 2.0:1; less than 1.5:1; or less than 1.0:1. In certain embodiments, the anti-C1q antibody binds to C1q with a binding stoichiometry in the range of 20:1 to 1.0:1 or less than 1.0:1. In certain embodiments, the anti-C1q antibody binds to C1q with a binding stoichiometry in the range of 6:1 to 1.0:1 or less than 1.0:1. In certain embodiments, the anti-C1q antibody binds to C1q with a binding stoichiometry ranging from 2.5:1 to 1.0:1 or less than 1.0:1. In some embodiments, the anti-C1q antibody inhibits the interaction between C1q and C1r, or between C1q and C1s, or between C1q and both C1r and C1s.In some embodiments, the anti-C1q antibody inhibits the interaction between C1q and C1r, between C1q and C1s, and / or between C1q and both C1r and C1s. In some embodiments, the anti-C1q antibody binds to the A chain of C1q. In other embodiments, the anti-C1q antibody binds to the B chain of C1q. In other embodiments, the anti-C1q antibody binds to the C chain of C1q. In some embodiments, the anti-C1q antibody binds to the A chain, the B chain, and / or the C chain of C1q. In some embodiments, the anti-C1q antibody binds to the globular domain of the A chain, the B chain, and / or the C chain of C1q. In other embodiments, the anti-C1q antibody binds to the collagen-like domain of the A chain, the B chain, and / or the C chain of C1q.

[0089] When an antibody of the present disclosure inhibits an interaction between two or more complement factors, e.g., the interaction between C1q and C1s or the interaction between C1q and C1r, the interaction that occurs in the presence of the antibody may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to a control in the absence of an antibody of the present disclosure. In some embodiments, an antibody of the present disclosure reduces the interaction between two or more complement factors by 50%, 60%, 70%, 80%, 90%, or 100%. In certain embodiments, the interaction that occurs in the presence of the antibody is reduced by an amount ranging from at least 30% to at least 99% compared to a control in the absence of an antibody of the present disclosure.

[0090] In some embodiments, an antibody of the present disclosure inhibits C2 or C4 cleavage by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or by an amount ranging from at least 30% to at least 99%, compared to a control in the absence of an antibody of the present disclosure. Methods for measuring C2 or C4 cleavage are well known in the art. The EC of an antibody of the present disclosure with respect to C2 or C4 cleavage can be measured using a ELISA.50 Values ​​can be less than 3 μg / ml; 2.5 μg / ml; 2.0 μg / ml; 1.5 μg / ml; 1.0 μg / ml; 0.5 μg / ml; 0.25 μg / ml; 0.1 μg / ml; 0.05 μg / ml. In some embodiments, the antibodies of the disclosure inhibit C2 or C4 cleavage at approximately equimolar concentrations of C1q and the respective anti-C1q antibody.

[0091] In some embodiments, an antibody of the disclosure inhibits autoantibody- and complement-dependent cytotoxicity (CDC) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or by an amount ranging from at least 30% to at least 99%, relative to a control in the absence of an antibody of the disclosure. 50 Values ​​may be less than 3 μg / ml; 2.5 μg / ml; 2.0 μg / ml; 1.5 μg / ml; 1.0 μg / ml; 0.5 μg / ml; 0.25 μg / ml; 0.1 μg / ml; 0.05 μg / ml.

[0092] In some embodiments, an antibody of the present disclosure inhibits complement-dependent cell-mediated cytotoxicity (CDCC) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or by an amount ranging from at least 30% to at least 99%, relative to a control in the absence of an antibody of the present disclosure. Methods for measuring CDCC are well known in the art. The EC of an antibody of the present disclosure with respect to CDCC inhibition is 50 Values ​​can be less than 3 μg / ml; 2.5 μg / ml; 2.0 μg / ml; 1.5 μg / ml; 1.0 μg / ml; 0.5 μg / ml; 0.25 μg / ml; 0.1 μg / ml; 0.05 μg / ml. In some embodiments, the antibodies of the disclosure inhibit CDCC but do not inhibit antibody-dependent cellular cytotoxicity (ADCC).

[0093] Humanized anti-complement C1q antibody The humanized antibodies of the present disclosure specifically bind to complement factor C1q and / or C1q protein in the C1 complex of the classical complement pathway. The humanized anti-C1q antibodies may specifically bind to human C1q, human and mouse C1q, rat C1q, or human C1q, mouse C1q, and rat C1q.

[0094] All sequences listed in the following 16 paragraphs are incorporated by reference from US Patent Application No. 14 / 933,517, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0095] In some embodiments, the human heavy chain constant region is a human IgG4 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% homology to SEQ ID NO: 47. The human IgG4 heavy chain constant region may comprise an Fc region with one or more modifications and / or amino acid substitutions according to Kabat numbering. In such cases, the Fc region comprises a leucine to glutamate amino acid substitution at position 248, which inhibits the Fc region from interacting with an Fc receptor. In some embodiments, the Fc region comprises a serine to proline amino acid substitution at position 241, which prevents arm switching in the antibody.

[0096] The amino acid sequence of the human IgG4 (S241P L248E) heavy chain constant domain is ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 47) is.

[0097] The antibody may comprise a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 31-34, or an amino acid sequence having at least about 90% identity to an amino acid sequence selected from any one of SEQ ID NOs: 31-34. In certain such embodiments, the light chain variable domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 35-38, or an amino acid sequence having at least about 90% identity to an amino acid sequence selected from any one of SEQ ID NOs: 35-38.

[0098] The amino acid sequence of the heavy chain variable domain variant 1 (VH1) is:

[0099] [ka] The hypervariable regions (HVRs) of VH1 are shown in bold and underlined text.

[0100] The amino acid sequence of the heavy chain variable domain variant 2 (VH2) is

[0101] [ka] The hypervariable regions (HVRs) of VH2 are shown in bold and underlined text.

[0102] The amino acid sequence of the heavy chain variable domain variant 3 (VH3) is

[0103] [ka] The hypervariable regions (HVRs) of VH3 are shown in bold and underlined text.

[0104] The amino acid sequence of the heavy chain variable domain variant 4 (VH4) is

[0105] [ka] The hypervariable region (HVR) of VH4 is shown in bold and underlined text.

[0106] The amino acid sequence of the kappa light chain variable domain variant 1 (Vκ1) is

[0107] [ka] The hypervariable regions (HVRs) of Vκ1 are shown in bold and underlined letters.

[0108] The amino acid sequence of the kappa light chain variable domain variant 2 (Vκ2) is

[0109] [ka] The hypervariable regions (HVRs) of Vκ2 are shown in bold and underlined letters.

[0110] The amino acid sequence of the kappa light chain variable domain variant 3 (Vκ3) is

[0111] [ka] The hypervariable regions (HVRs) of Vκ3 are shown in bold and underlined letters.

[0112] The amino acid sequence of the kappa light chain variable domain variant 4 (Vκ4) is

[0113] [ka] The hypervariable region (HVR) of Vκ4 is shown in bold and underlined letters.

[0114] The antibody may comprise a light chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NOs: 35-38, while retaining HVR-L1 RASKSINKYLA (SEQ ID NO: 5), HVR-L2 SGSTLQS (SEQ ID NO: 6), and HVR-L3 QQHNEYPLT (SEQ ID NO: 7). The antibody may comprise a heavy chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NOs: 31-34, while preferably retaining HVR-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO: 10), and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0115] In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 35 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 31. In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 36 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 32. In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 37 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 38 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 34.

[0116] In some embodiments, a humanized anti-C1q antibody of the present disclosure comprises a heavy chain variable region containing a Fab region and a heavy chain constant region containing an Fc region, wherein the Fab region specifically binds to a C1q protein of the present disclosure, while the Fc region is incapable of binding to a C1q protein. In some embodiments, the Fc region is derived from a human IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fc region is incapable of inducing complement activity and / or incapable of inducing antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the Fc region comprises one or more modifications, including, but not limited to, an amino acid substitution. In certain embodiments, the Fc region of a humanized anti-C1q antibody of the present disclosure comprises an amino acid substitution at position 248 according to the Kabat numbering convention, or a position corresponding to position 248 according to the Kabat numbering convention, and / or at position 241 according to the Kabat numbering convention, or a position corresponding to position 241 according to the Kabat numbering convention. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 prevents the Fc region from interacting with an Fc receptor. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 is a leucine to glutamate amino acid substitution. In some embodiments, the amino acid substitution at position 241 or a position corresponding to position 241 prevents arm switching in the antibody. In some embodiments, the amino acid substitution at position 241 or a position corresponding to position 241 is a serine to proline amino acid substitution. In certain embodiments, the Fc region of a humanized anti-C1q antibody of the present disclosure comprises the amino acid sequence of SEQ ID NO:47, or an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity to the amino acid sequence of SEQ ID NO:47.

[0117] Anti-C1q Fab fragment Before the advent of recombinant DNA technology, proteolytic enzymes (proteases) that cleave polypeptide sequences were used to break down the structure of antibody molecules and determine which parts of the molecule are responsible for their various functions. Limited digestion with the protease papain cleaves antibody molecules into three fragments. Two of the fragments, known as Fab fragments, are identical and contain antigen-binding activity. The Fab fragments correspond to two identical arms of the antibody molecule, each of which contains the V-terminal end of the heavy chain. H and C H The Fab fragment consists of an intact light chain paired with an Fc domain. The other fragment, while not containing antigen-binding activity, was initially observed to be readily crystallizable, and for this reason was named the Fc fragment (fragment crystallizable). When Fab molecules were compared with IgG molecules, Fabs were found to be superior to IgG for certain in vivo applications due to their higher mobility and tissue penetration capacity, their reduced circulating half-life, their ability to bind antigen monovalently without mediating antibody effector functions, and their lower immunogenicity.

[0118] Fab molecules contain the constant domain C H 2 and C H It is an artificial approximately 50 kDa fragment of an Ig molecule with a heavy chain shortened by 3. Two heterophils (V L -V H and C L -C H 1) Domain interactions underlie the structure of the two chains of the Fab molecule, which is C L and C H Fab and IgG are further stabilized by disulfide bridges between the V and V domains. Fab and IgG have six complementarity-determining regions (CDRs), three of which are V and V domains. L and V H The CDRs define the hypervariable antigen-binding site of an antibody. The highest sequence variation is found in LCDR3 and HCDR3, which in the natural immune system are V and V, respectively. L and J L Gene or V H , DH and J H They are generated by genetic rearrangement. LCDR3 and HCDR3 typically form the core of the antigen-binding site. The conserved regions connecting and displaying the six CDRs are called framework regions. In the three-dimensional structure of the variable domain, the framework regions form a sandwich of two opposing antiparallel β-sheets linked on the outside by the hypervariable CDR loops and on the inside by conserved disulfide bridges. This unique combination of stability and versatility of the antigen-binding sites of Fab and IgG underscores their success in clinical practice for the diagnosis, monitoring, prevention, and treatment of disease.

[0119] All anti-C1q antibody Fab fragment sequences are incorporated by reference from US Patent Application No. 15 / 360,549, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0120] In certain embodiments, the present disclosure provides a method for the production of a heavy chain (V H / C H 1) and light chain (V L / C L ), wherein the anti-C1q antibody Fab fragment binds to the C1q protein and comprises six complementarity-determining regions (CDRs) (three of which are V L and V H The heavy chain of the antibody Fab fragment is cleaved after the first heavy chain domain of IgG1 (SEQ ID NO: 39) and comprises the following amino acid sequence:

[0121] [ka]

[0122] The complementarity determining regions (CDRs) of SEQ ID NO: 1 are shown in bold and underlined text.

[0123] The light chain domain of the antibody Fab fragment comprises the following amino acid sequence (SEQ ID NO: 40):

[0124] [ka]

[0125] The complementarity determining regions (CDRs) of SEQ ID NO:2 are shown in bold and underlined text.

[0126] Anti-complement C1s antibody Suitable inhibitors include antibodies that bind to the complement C1s protein (i.e., anti-complement C1s antibodies, also referred to herein as anti-C1s antibodies and C1s antibodies) and nucleic acid molecules encoding such antibodies. Complement C1s is an attractive target because it is upstream in the complement cascade and has a narrow range of substrate specificity. Furthermore, it is possible to obtain antibodies (e.g., but not limited to, monoclonal antibodies) that specifically bind to the activated form of C1s.

[0127] All sequences listed in the following two paragraphs are incorporated by reference from US Patent Application No. 14 / 890,811, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0128] In certain aspects, disclosed herein are methods of administering an anti-C1s antibody. The antibody can be a murine antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3, and the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3 of murine anti-human C1s monoclonal antibody 5A1 produced by the hybridoma cell line deposited with the ATCC on 5 / 15 / 2013 or its progeny (ATCC Accession No. PTA-120351). In another embodiment, the light chain variable domain comprises HVR-L1, HVR-L2, and HVR-L3, and the heavy chain variable domain comprises HVR-H1, HVR-H2, and HVR-H3 of the mouse anti-human C1s monoclonal antibody 5C12 produced by the hybridoma cell line deposited with the ATCC on 5 / 15 / 2013 or its progeny (ATCC Accession No. PTA-120352).

[0129] In some embodiments, the antibody specifically binds to C1s or C1s proenzyme and inhibits its biological activity, e.g., C1s binding to C1q, C1s binding to C1r, or C2 or C4. The biological activity can be the proteolytic activity of C1s, conversion of the C1s proenzyme to an active protease, or proteolytic cleavage of C2 or C4. In certain embodiments, the biological activity is activation of the classical complement activation pathway, activation of antibody and complement-dependent cytotoxicity, or C1F hemolysis.

[0130] All sequences in the following 62 paragraphs are incorporated by reference from Van Vlasselaer, US Patent No. 8,877,197, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0131] Disclosed herein are methods of administering a humanized monoclonal antibody that specifically binds to an epitope within a region encompassing domains IV and V of complement component C1s. In some cases, the antibody inhibits binding of C1s to complement component 4 (C4) and / or does not inhibit the protease activity of C1s. In some embodiments, the method comprises administering a humanized monoclonal antibody that binds with high avidity to complement component C1s in the C1 complex.

[0132] Disclosed herein are methods of administering an anti-C1s antibody having one or more complementarity-determining regions (CDRs) of an antibody light chain variable region comprising the amino acid sequence SEQ ID NO: 57 and / or one or more CDRs of an antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO: 58. The anti-C1s antibody can bind to human or rat complement C1s protein. In some embodiments, the anti-C1s antibody inhibits cleavage of at least one substrate cleaved by the complement C1s protein.

[0133] In certain embodiments, the antibody comprises a) a complementarity determining region (CDR) having an amino acid sequence selected from SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56; and / or b) a CDR having an amino acid sequence selected from SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:53, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0134] The antibody may comprise a CDR-L1 having the amino acid sequence SEQ ID NO:51, a CDR-L2 having the amino acid sequence SEQ ID NO:52, a CDR-L3 having the amino acid sequence SEQ ID NO:53, a CDR-H1 having the amino acid sequence SEQ ID NO:54, a CDR-H2 having the amino acid sequence SEQ ID NO:55, and a CDR-H3 having the amino acid sequence SEQ ID NO:56.

[0135] In other embodiments, the antibody may comprise a light chain CDR of a variable region having the amino acid sequence of SEQ ID NO:67 and / or a heavy chain CDR of a variable region having the amino acid sequence of SEQ ID NO:68.

[0136] The antibody may be a humanized antibody that specifically binds to complement component C1s, and that competes for binding to an epitope with an antibody comprising one or more CDRs of an antibody light chain variable region comprising the amino acid sequence SEQ ID NO:57 or SEQ ID NO:67 and / or one or more CDRs of an antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO:58 or SEQ ID NO:68.

[0137] In another example, the antibody may be a humanized antibody that specifically binds to complement C1s, the antibody being selected from: 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 SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56; 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 SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:53, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66. In some cases, the antibody competes for binding to the epitope with an antibody comprising heavy and light chain CDRs comprising: a) SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:69, SEQ ID NO:55, and SEQ ID NO:56; or b) SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:53, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0138] An antibody may comprise a light chain region and a heavy chain region that are present on separate polypeptides. An antibody may comprise an Fc region.

[0139] Disclosed herein is an anti-C1s antibody comprising a light chain variable region having an amino acid sequence 90% identical to amino acid sequence SEQ ID NO:57, and a heavy chain variable region comprising an amino acid sequence 90% identical to amino acid sequence SEQ ID NO:58.

[0140] The anti-C1s antibody may be selected from an antigen-binding fragment, an Ig monomer, a Fab fragment, a F(ab')2 fragment, a Fd fragment, an scFv, a scAb, a dAb, an Fv, a single-domain heavy chain antibody, a single-domain light chain antibody, a monospecific antibody, a bispecific antibody, or a multispecific antibody.

[0141] Disclosed herein are methods of administering an antibody that competes for binding to the epitope bound by antibody IPN003 (also referred to herein as "IPN-M34" or "M34" or "TNT003"), e.g., an antibody comprising the variable domain of antibody IPN003, such as antibody IPN003.

[0142] In some embodiments, the method includes administering an antibody that specifically binds to an epitope within the complement C1s protein. In some embodiments, the isolated anti-C1s antibody binds to the activated C1s protein. In some embodiments, the isolated anti-C1s antibody binds to the inactive form of C1s. In other examples, the isolated anti-C1s antibody binds to both the activated C1s protein and the inactive form of C1s.

[0143] In some embodiments, the method includes administering a monoclonal antibody that inhibits cleavage of C4, wherein the isolated monoclonal antibody does not inhibit cleavage of C2. In some embodiments, the method includes administering a monoclonal antibody that inhibits cleavage of C2, wherein the isolated monoclonal antibody does not inhibit cleavage of C4. In some cases, the isolated monoclonal antibody is humanized. In some cases, the antibody inhibits a component of the classical complement pathway. In some cases, the component of the classical complement pathway inhibited by the antibody is C1s. The present disclosure also provides a method of treating a complement-mediated disease or disorder by administering to an individual in need thereof an isolated monoclonal antibody that inhibits cleavage of C4, or a pharmaceutical composition comprising the isolated monoclonal antibody, wherein the isolated monoclonal antibody does not inhibit cleavage of C2.

[0144] In some embodiments, the method includes administering a monoclonal antibody that inhibits cleavage of C2 or C4 by C1s, i.e., inhibits C1s-mediated proteolytic cleavage of C2 or C4. In some cases, the monoclonal antibody is humanized. In some cases, the antibody inhibits cleavage of C2 or C4 by C1s by inhibiting binding of C2 or C4 to C1s; for example, in some cases, the antibody inhibits C1s-mediated cleavage of C2 or C4 by inhibiting binding of C2 or C4 to the C2 or C4 binding site of C1s. Thus, in some cases, the antibody functions as a competitive inhibitor. The present disclosure also provides for the treatment of blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia, and the like) by administering to an individual in need thereof an isolated monoclonal antibody that inhibits cleavage of C2 or C4 by C1s, i.e., inhibits C1s-mediated proteolytic cleavage of C2 or C4. and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin).

[0145] In some embodiments, the method includes administering a monoclonal antibody that inhibits cleavage of C4 by C1s, where the antibody does not inhibit cleavage of complement component C2 by C1s; i.e., the antibody inhibits C1s-mediated cleavage of C4 but does not inhibit C1s-mediated cleavage of C2. In some cases, the monoclonal antibody is humanized. In some cases, the monoclonal antibody inhibits binding of C4 to C1s but does not inhibit binding of C2 to C1s. In some embodiments, the method includes treating a complement-mediated disease or disorder by administering to an individual in need thereof an isolated monoclonal antibody that inhibits cleavage of C4 by C1s, where the antibody does not inhibit cleavage of complement component C2 by C1s; i.e., the antibody inhibits C1s-mediated cleavage of C4 but does not inhibit C1s-mediated cleavage of C2. In some embodiments of the method, the antibody is humanized.

[0146] In some embodiments, the method comprises administering a humanized monoclonal antibody that specifically binds to an epitope within a region encompassing domains IV and V of C1s. For example, the 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: 70. In some cases, the 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: 70 and inhibits binding of C4 to C1s. In some embodiments, the method comprises treating a complement-mediated disease or disorder by administering to an individual in need thereof a 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: 70 and inhibits binding of C4 to C1s.

[0147] In some embodiments, the method includes administering a humanized monoclonal antibody that specifically binds to a conformational epitope within a region encompassing domains IV and V of C1s, for example, a 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: 70. In some cases, the humanized monoclonal antibody 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: 70 and inhibits binding of C4 to C1s. In some embodiments, the method relates to the treatment of a blood disorder (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (AIP). and a 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: 70 and inhibits binding of C4 to C1s.

[0148] In some embodiments, the method includes administering a monoclonal antibody that binds to complement component C1s in the C1 complex. The C1 complex is composed of six molecules of C1q, two molecules of C1r, and two molecules of C1s. In some cases, the monoclonal antibody is humanized. Thus, in some cases, the monoclonal antibody binds to complement component C1s in the C1 complex. In some cases, the antibody binds to C1s present in the C1 complex with high avidity.

[0149] In some embodiments, an anti-C1s antibody (e.g., a subject antibody that specifically binds to an epitope in the complement C1s protein) comprises: a) a light chain region comprising one, two, or three VL CDRs of the IPN003 antibody; and b) a heavy chain region comprising one, two, or three VH CDRs of the IPN003 antibody, wherein the VH and VL CDRs are as defined by Kabat (Kabat 1991).

[0150] In other embodiments, an anti-C1s antibody (e.g., a subject antibody that specifically binds to an epitope in the complement C1s protein) comprises: a) a light chain region comprising one, two, or three VL CDRs of the IPN003 antibody; and b) a heavy chain region comprising one, two, or three VH CDRs of the IPN003 antibody, wherein the VH and VL CDRs are as defined by Chothia (Chothia 1987).

[0151] In some embodiments, an anti-C1s antibody (e.g., a subject antibody that specifically binds to an epitope in the complement C1s protein) comprises: a) a light chain region comprising one, two, or three CDRs selected from SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53; and b) a heavy chain region comprising one, two, or three CDRs selected from SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56. In some of these embodiments, the anti-C1s antibody comprises humanized VH and / or VL framework regions.

[0152] SEQ ID NO: 51: SSVSSSYLHWYQ; SEQ ID NO: 52: STSNLASGVP; SEQ ID NO: 53: HQYYRLPPIT; SEQ ID NO:54: GFTFSNYAMSWV; SEQ ID NO: 55:ISSGGSHTYY; SEQ ID NO: 56: ARLFTGGYAMDY.

[0153] In some embodiments, the anti-C1s antibody comprises a CDR having an amino acid sequence selected from SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.

[0154] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising the amino acid sequences SEQ ID NO:51, SEQ ID NO:52, and SEQ ID NO:53.

[0155] In some embodiments, the anti-C1s antibody comprises a heavy chain variable region comprising the amino acid sequences SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.

[0156] In some embodiments, the anti-C1s antibody comprises a CDR-L1 having the amino acid sequence SEQ ID NO:51, a CDR-L2 having the amino acid sequence SEQ ID NO:52, a CDR-L3 having the amino acid sequence SEQ ID NO:53, a CDR-H1 having the amino acid sequence SEQ ID NO:54, a CDR-H2 having the amino acid sequence SEQ ID NO:55, and a CDR-H3 having the amino acid sequence SEQ ID NO:56.

[0157] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:57.

[0158] SEQ ID NO:57: DIVMTQTTAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTFYSLTISSMEAEDDATYYCHQYYRLPPITFGAGTKLELK.

[0159] In some embodiments, the anti-C1s antibody comprises a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:58.

[0160] SEQ ID NO:58: QVKLEESGGALVKPGGSLKLSCAASGFTFSNYAMSWVRQIPEKRLEWVATISSGGSHTYYLDSVKGRFTISRDNARDTLYLQMSSLRSEDTALYYCARLFTGYAMDYWGQGTSVT.

[0161] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence SEQ ID NO:57.

[0162] In some embodiments, the anti-C1s antibody comprises a heavy chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence SEQ ID NO:58.

[0163] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising the amino acid sequence SEQ ID NO:57.

[0164] In some embodiments, the anti-C1s antibody comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO:58.

[0165] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence SEQ ID NO:57 and a heavy chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence SEQ ID NO:58.

[0166] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising the amino acid sequence SEQ ID NO:57 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:58.

[0167] In some embodiments, the anti-C1s antibody specifically binds to an epitope within the complement C1s protein, and the antibody competes for binding to the epitope with an antibody comprising a light chain CDR of an antibody light chain variable region comprising the amino acid sequence SEQ ID NO: 57 and a heavy chain CDR of an antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO: 58.

[0168] In some embodiments, the anti-C1s antibody comprises a light chain CDR of an antibody light chain variable region comprising the amino acid sequence SEQ ID NO:57 and a heavy chain CDR of an antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO:58.

[0169] In some embodiments, an anti-C1s antibody (e.g., a subject antibody that specifically binds to an epitope in the complement C1s protein) comprises: a) a light chain region comprising one, two, or three CDRs selected from SEQ ID NO: 62, SEQ ID NO: 63, and SEQ ID NO: 53; and b) a heavy chain region comprising one, two, or three CDRs selected from SEQ ID NO: 64, SEQ ID NO: 65, and SEQ ID NO: 66.

[0170] SEQ ID NO: 62: TASSSVSSSYLH; SEQ ID NO: 63: STSNLAS; SEQ ID NO: 53: HQYYRLPPIT; SEQ ID NO: 64: NYAMS; SEQ ID NO: 65: TISSGGSHTYYLDSVKG; SEQ ID NO: 66: LFTGHAMDY

[0171] In some embodiments, the anti-C1s antibody comprises a CDR having an amino acid sequence selected from SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:53, SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0172] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising the amino acid sequences SEQ ID NO:62, SEQ ID NO:63, and SEQ ID NO:53.

[0173] In some embodiments, the anti-C1s antibody comprises a heavy chain variable region comprising the amino acid sequences SEQ ID NO:64, SEQ ID NO:65, and SEQ ID NO:66.

[0174] In some embodiments, the anti-C1s antibody comprises a CDR-L1 having the amino acid sequence SEQ ID NO: 62, a CDR-L2 having the amino acid sequence SEQ ID NO: 63, a CDR-L3 having the amino acid sequence SEQ ID NO: 53, a CDR-H1 having the amino acid sequence SEQ ID NO: 64, a CDR-H2 having the amino acid sequence SEQ ID NO: 65, and a CDR-H3 having the amino acid sequence SEQ ID NO: 66.

[0175] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:67.

[0176] SEQ ID NO:67: QIVLTQSPAIMSASLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTFYSLTISSMEAEDDATYYCHQYYRLPPITFGAGTKLELK.

[0177] In some embodiments, the anti-C1s antibody comprises a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:68.

[0178] SEQ ID NO:68: EVMLVESGGALVKPGGSLKLSCAASGFTFSNYAMSWVRQIPEKRLEWVATISSGGSHTYYLDSVKGRFTISRDNARDTLYLQMSSLRSEDTALYYCARLFTGYAMDYWGQGTSVTVSS.

[0179] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence SEQ ID NO:67.

[0180] In some embodiments, the anti-C1s antibody comprises a heavy chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence SEQ ID NO:68.

[0181] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising the amino acid sequence SEQ ID NO:67.

[0182] In some embodiments, the anti-C1s antibody comprises a heavy chain variable region comprising the amino acid sequence SEQ ID NO:68.

[0183] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence SEQ ID NO:67 and a heavy chain variable region comprising an amino acid sequence that is 90% identical to the amino acid sequence SEQ ID NO:68.

[0184] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 95% identical to the amino acid sequence SEQ ID NO:67 and a heavy chain variable region comprising an amino acid sequence that is 95% identical to the amino acid sequence SEQ ID NO:68.

[0185] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising the amino acid sequence SEQ ID NO:67 and a heavy chain variable region comprising the amino acid sequence SEQ ID NO:68.

[0186] In some embodiments, the anti-C1s antibody specifically binds to an epitope within the complement C1s protein, and the antibody competes for binding to the epitope with an antibody comprising a light chain CDR of an antibody light chain variable region comprising the amino acid sequence SEQ ID NO: 67 and a heavy chain CDR of an antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO: 68.

[0187] In some embodiments, the anti-C1s antibody comprises a light chain CDR of an antibody light chain variable region comprising the amino acid sequence SEQ ID NO:67 and a heavy chain CDR of an antibody heavy chain variable region comprising the amino acid sequence SEQ ID NO:68.

[0188] In some embodiments, the anti-C1s antibody comprises a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:67.

[0189] In some embodiments, the anti-C1s antibody comprises a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence set forth in SEQ ID NO:68.

[0190] The anti-C1s antibody may comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 79 and illustrated in Figure 2 (VH variant 1).

[0191] The anti-C1s antibody may comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 80 and depicted in Figure 3 (VH variant 2).

[0192] The anti-C1s antibody may comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 81 and illustrated in Figure 4 (VH variant 3).

[0193] The anti-C1s antibody may comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 82 and illustrated in Figure 5 (VH variant 4).

[0194] The anti-C1s antibody may comprise a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 83 and illustrated in Figure 6 (VK variant 1).

[0195] The anti-C1s antibody may comprise a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 84 and illustrated in Figure 7 (VK variant 2).

[0196] The anti-C1s antibody may comprise a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 85 and illustrated in Figure 8 (VK variant 3).

[0197] The anti-C1s antibody may comprise a heavy chain variable region that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the framework (FR) amino acid substitutions relative to the IPN003 parent antibody FR amino acid sequence shown in Table 3 (Figure 9).

[0198] definition As used herein, "a" or "an" can mean one or more. As used herein in the claim(s), when used in conjunction with the term "comprising," the term "a" or "an" can mean one or more. For example, a reference to an "antibody" is a reference to one to many antibodies. As used herein, "another" can mean at least a second or more.

[0199] As used herein, administration "in conjunction with" another compound or composition includes simultaneous administration and / or administration at different times. Concurrent administration also encompasses administration as a co-formulation or as separate compositions, including using different dosing frequencies or intervals, and the same or different routes of administration.

[0200] "Complement-mediated blood disorders" are disorders of vascular compartments or highly vascularized tissues caused by circulating C1q and complement activation. Complement activation can be initiated via the classical pathway. The classical pathway can be activated by direct binding of the complement protein C1q to surface-bound antibodies or patches of surface proteins.

[0201] The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody." The term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, antibody fragments, and antibody derivatives, so long as they exhibit biological activity.

[0202] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. H and V L When these amino acids pair together, they form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0203] Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domain. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. Five classes of immunoglobulins exist: IgA, IgD, IgE, IgG, and IgM (with heavy chains designated alpha ("α"), delta ("δ"), epsilon ("ε"), gamma ("γ"), and mu ("μ"), respectively). The gamma and alpha classes are further divided into subclasses (isotypes) based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and generally described in, e.g., Abbas et al., Cellular and Molecular Immunology, 4 th This is described in ed. (WBSaunders Co., 2000).

[0204] "Full-length antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, comprising two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ) followed by several constant domains. Each light chain has a variable domain (V) at one end. L ) at its other end and a constant domain, with the light-chain constant domain aligned with the first constant domain of the heavy chain and the light-chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain.

[0205] An "isolated" molecule or cell is a molecule or cell that has been identified and separated from at least one contaminating molecule or cell with which it is normally associated in the environment in which it is produced. Preferably, an isolated molecule or cell is free from association with all components associated with the environment in which it is produced. An isolated molecule or cell is in a form other than the form or context in which it is found in nature. As such, an isolated molecule is distinguished from molecules naturally occurring in a cell; an isolated cell is distinguished from cells naturally occurring in a tissue, organ, or individual. In some embodiments, the isolated molecule is an anti-C1s, anti-C1q, or anti-C1r antibody of the present disclosure. In other embodiments, the isolated cell is a host cell or hybridoma cell that produces an anti-C1s, anti-C1q, or anti-C1r antibody of the present disclosure.

[0206] An "isolated" antibody is one that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free of association with all other contaminating components from its production environment. Contaminating components from its production environment, such as those resulting from recombinantly transfected cells, are substances that would typically interfere with research, diagnostic, or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain preferred embodiments, the polypeptide will be purified (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, of the antibody, as determined, for example, by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue, or preferably silver stain. Isolated antibodies include antibodies in situ within recombinant T cells, since at least one component of the antibody's natural environment will be absent. Ordinarily, however, an isolated polypeptide or antibody will be prepared by a process that includes at least one purification step.

[0207] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy and light chains are respectively referred to as "V H " and "V L These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding sites.

[0208] The term "variable" refers to the fact that certain segments of variable domains vary significantly in sequence among different antibodies. V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in both the light- and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy- and light-chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration, connected by three HVRs that form loops that connect, and in some cases form part of, the beta-sheet structure. The HVRs within each chain are held in close proximity by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0209] As used herein, the term "CDR" or "complementarity-determining region" is intended to mean the noncontiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996) (the definitions include overlapping or subsets of amino acid residues when compared against each other). Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein.

[0210] As used herein, the terms "CDR-L1," "CDR-L2," and "CDR-L3" refer to the first, second, and third CDRs, respectively, in a 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, in the variable region of either chain.

[0211] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies of the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are typically synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present disclosure can be produced using, for example, hybridoma methods (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2d ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display techniques (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al. al.,J.Mol.Biol.222:581-597(1992);Sidhu et al.,J.Mol.Biol.338(2):299-310(2004);Lee et al.,J.Mol.Biol.340(5):1073-1093(2004);Fellouse,Proc.Nat'l Acad.Sci.USA 101(34):12467-472 (2004); and Lee et al., J. Immunol Methods 284(1-2):119-132 (2004)), and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998 / 24893; WO 1996 / 34096; WO 1996 / 33735; WO 1991 / 10741; Jakobovits et al., Proc. Nat'l Acad. Sci. USA 90:2551 (1993); Jakobovits et al., Nature 362:255-258 (1993); Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-813 (1994); Fishwild et al., Nature Biotechnol. 14:845-851 (1996); Neuberger, Nature Biotechnol. 14:826 (1996); and Lonberg and These antibodies can be produced by a variety of techniques, including immunoglobulin A (see Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0212] The terms "full length antibody," "intact antibody," and "complete antibody" are used interchangeably to refer to an antibody in substantially intact form, as opposed to an antibody fragment or antibody derivative. Specifically, complete antibodies include those having heavy and light chains, including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

[0213] An "antibody fragment" or "antigen-binding fragment" or "functional fragment" of an antibody comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody or the F region of the antibody that retains or has altered FcR binding ability. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; and linear antibodies (see U.S. Patent 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)). Additional examples of antibody fragments include antibody derivatives, such as single-chain antibody molecules formed from antibody fragments, monovalent antibodies, and multispecific antibodies.

[0214] An "antibody derivative" is any construct that contains the antigen-binding region of an antibody. Examples of antibody derivatives include single-chain antibody molecules formed from antibody fragments, monovalent antibodies, and multispecific antibodies.

[0215] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments and one residual "Fc" fragment, a designation reflecting its ability to crystallize readily. The Fab fragment contains the entire light chain plus the variable region domain of the heavy chain (V H ), and the first constant domain of one heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. The Fab' fragment is C H F(ab')2 antibody fragments differ from Fab fragments by having several additional residues at the carboxy terminus of one domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0216] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0217] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain and includes native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, an intact antibody composition may include an antibody population with all K447 residues removed, an antibody population lacking the removed K447 residue, and an antibody population having a mixture of antibodies with and without the K447 residue. Native-sequence Fc regions suitable for use in the antibodies of the present disclosure include human IgG1, IgG2, IgG3, and IgG4.

[0218] A "native sequence Fc region" comprises an amino acid sequence identical to that of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes), native sequence human IgG2 Fc regions, native sequence human IgG3 Fc regions, and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0219] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide. A variant Fc region herein preferably retains at least about 80% homology with a native-sequence Fc region and / or the Fc region of the parent polypeptide, and most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0220] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native-sequence human FcR. Furthermore, a preferred FcR binds IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif ("ITIM") in its cytoplasmic domain. (See, e.g., M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. FcRs may increase the serum half-life of antibodies.

[0221] The in vivo FcRn binding and serum half-life of human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which polypeptides having variant Fc regions are administered. WO 2004 / 42072 (Presta) describes antibody variants with improved or reduced FcR binding. See, e.g., Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).

[0222] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. The folding of these two domains generates six hypervariable loops (three loops each from the H chain and L chain) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to an antigen, albeit with lower affinity than the entire binding site.

[0223] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the VH and VL antibody domains joined in a single polypeptide chain. Preferably, the sFv polypeptide comprises a VH and VL antibody domain. H and V L The sFv further comprises a polypeptide linker between the domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0224] The term "diabody" refers to a diabody that combines V domains to achieve inter-chain but not intra-chain V domain pairing, thereby resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. H Domains and V L This refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. Bispecific diabodies are small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. H and V LDiabodies are heterodimers of two "crossover" sFv fragments in which the domains are present on different polypeptide chains. Diabodies are described in more detail in, for example, EP 404,097; WO 1993 / 011161; WO / 2009 / 121948; WO / 2014 / 191493; Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48 (1993).

[0225] As used herein, "chimeric antibody" refers to antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies, in which the antigen-binding region of the antibody is derived from an antibody generated, for example, by immunizing macaque monkeys with the antigen of interest. As used herein, "humanized antibodies" are a subset of "chimeric antibodies."

[0226] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FRs typically will be no more than six in the H chain and no more than three in the L chain. The humanized antibody also optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0227] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomies, that have been engineered to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See also Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies produced by human B cell hybridoma technology.

[0228] As used herein, the terms "hypervariable region," "HVR," or "HV" refer to the region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Generally, antibodies contain six HVRs: three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). In natural antibodies, H3 and L3 exhibit the highest diversity among these six HVRs, and H3 in particular is thought to play a unique role in conferring superior specificity to antibodies. See, e.g., Xu et al., Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0229] Several HVR delineations are used and encompassed herein. Kabat complementarity-determining regions (CDRs), HVRs, are based on sequence variability and are the most commonly used (Kabat et al., supra). Chothia, instead, refer to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are described below.

[0230] [Table 2]

[0231] HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65 or 49-65 (preferred embodiment) (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these extended HVR definitions.

[0232] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0233] The phrases "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat," and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or HVRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues can be determined for a given antibody by matching the antibody's sequence with the "standard" Kabat-numbered sequence at the regions of homology.

[0234] The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise stated herein, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system (see, e.g., U.S. Patent Publication No. 2010-280227).

[0235] An "acceptor human framework," as used herein, is a framework that comprises the amino acid sequence of a VL framework or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence as that of the human immunoglobulin framework or the human consensus framework, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. When pre-existing amino acid changes are present in the VH, preferably those changes are present at only three, two, or one of positions 71H, 73H, and 78H; for example, the amino acid residues at those positions may be 71A, 73T, and / or 78A. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0236] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is made from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include for VL, the subgroup can be subgroup kappa I, kappa II, kappa III, or kappa IV as in Kabat et al., supra. Additionally, for VH, the subgroup can be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.

[0237] An "amino acid modification" at a specified position refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent" to a specified residue means an insertion within one to two residues thereof. The insertion may be on the N-terminal or C-terminal side of the specified residue. A preferred amino acid modification herein is a substitution.

[0238] An "affinity matured" antibody is one that contains one or more changes in one or more HVRs thereof that result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody that does not possess those change(s). In some embodiments, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are produced by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and / or framework residues is described, for example, in Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0239] As used herein, the terms "specifically recognize" or "specifically bind" refer to a measurable and reproducible interaction, such as attraction or binding, between a target and an antibody that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically or preferentially binds to a target or epitope is one that binds to this target or epitope with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other targets or other epitopes of targets. For example, it is understood that an antibody (or moiety) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. An antibody that specifically binds to a target has at least about 10 3 M -1 or 10 4 M -1 , sometimes about 10 5 M -1 or 10 6 M -1 , and in other cases, about 10 6 M -1 or 10 7 M -1 , about 10 8 M -1 ~10 9 M -1 , or about 10 10 M -1 ~10 11 M -1 The antibody may have a binding constant of 100 uA or higher. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0240] "Identity," as used herein, indicates that the amino acid residue at any particular position in the aligned sequences is the same between the sequences. "Similarity," as used herein, indicates that the amino acid residue at any particular position in the aligned sequences is of a similar type between the sequences. For example, leucine can be substituted for isoleucine or valine. Other amino acids that can often be substituted for one another include: - phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains); - lysine, arginine and histidine (amino acids with basic side chains); - aspartate and glutamate (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and -cysteine ​​and methionine (amino acids with sulfur-containing side chains) These include, but are not limited to:

[0241] The degree of identity and similarity can be readily calculated (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing, Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991).

[0242] As used herein, an "interaction" between a complement protein and a second protein includes, but is not limited to, protein-protein interactions, physical interactions, chemical interactions, bonds, covalent bonds, and ionic bonds. As used herein, an antibody "inhibits the interaction" between two proteins if the antibody disrupts, reduces, or completely eliminates the interaction between the two proteins. An antibody of the present disclosure, or a fragment thereof, "inhibits the interaction" between two proteins if the antibody or a fragment thereof binds to one of the two proteins.

[0243] A "blocking," "antagonist," "inhibitory," or "neutralizing" antibody is an antibody that inhibits or reduces one or more biological activities of the antigen to which it binds, e.g., its interaction with one or more proteins. In some embodiments, a blocking, antagonist, inhibitory, or "neutralizing" antibody substantially or completely inhibits one or more biological activities or interactions of an antigen.

[0244] The term "inhibitor" refers to a compound that has the ability to inhibit the biological function of a target biomolecule, such as mRNA or protein, whether by reducing the activity or expression of the target biomolecule. An inhibitor can be an antibody, a small molecule, or a nucleic acid molecule. The term "antagonist" refers to a compound that binds to a receptor and blocks or attenuates the biological response of the receptor. The term "inhibitor" can also refer to an "antagonist."

[0245] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype.

[0246] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two substances (e.g., an antibody and an antigen), expressed as the dissociation constant (KD). The affinity can be at least 1-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 20-fold higher, at least 30-fold higher, at least 40-fold higher, at least 50-fold higher, at least 60-fold higher, at least 70-fold higher, at least 80-fold higher, at least 90-fold higher, at least 100-fold higher, or at least 1,000-fold higher, or more, than the affinity of the antibody for an unrelated amino acid sequence. The affinity of an antibody for a target protein can be, for example, about 100 nanomolar (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolar (fM) or greater. As used herein, the term "avidity" refers to the resistance of a complex of two or more substances to dissociation after dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0247] The term "binding" refers to a direct association between two molecules, e.g., by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bonding interactions, including interactions such as salt bridges and water bridges. For example, a subject anti-C1s antibody specifically binds to an epitope within the complement C1s protein. "Specific binding" refers to binding of at least about 10 -7 M or more, e.g., 5 x 10 -7 M, 10 -8 M, 5 x 10 -8 "Non-specific binding" refers to binding with an affinity of about 10 M or greater. -7 Affinity less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 It refers to binding with an affinity such as M.

[0248] The term "k" on" as used herein is intended to refer to the rate constant for the association of an antibody to an antigen.

[0249] The term "k" off ", as used herein, is intended to refer to the rate constant for dissociation of an antibody from the antibody / antigen complex.

[0250] The term “K D ", as used herein, is intended to refer to the equilibrium dissociation constant of an antibody-antigen interaction.

[0251] As used herein, with respect to peptide, polypeptide, or antibody sequences, "percent (%) amino acid sequence identity" and "homology" refer to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art necessary to achieve maximal alignment over the entire length of the sequences being compared.

[0252] A "biological sample" encompasses a variety of sample types obtained from an individual and can be used in diagnostic or monitoring assays. The definition includes blood and other liquid samples of biological origin, solid tissue samples, such as biopsies or tissue cultures, or cells derived therefrom and their progeny. The definition also includes samples that have been manipulated in any way after their procurement, for example, by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term "biological sample" encompasses clinical samples, and also includes cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, 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.

[0253] "Blood space," as that term is used herein, refers to the contents of a subject's cardiovascular system, including serum, platelets, endothelial cells, blood cells and other hematopoietic cells, and other substances that naturally flow through a subject's circulatory system. Targeting the blood space can have an effect on highly vascular tissues, such as the kidney, alveoli, capillary beds, or glomeruli.

[0254] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it is produced. Preferably, an isolated nucleic acid is free from association with all components associated with the environment in which it is produced. Isolated nucleic acid molecules encoding the polypeptides and antibodies herein are in a form other than in the form or setting in which they are found in nature. As such, isolated nucleic acid molecules are distinguished from nucleic acids encoding any polypeptides and antibodies herein that naturally occur in a cell.

[0255] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." Typically, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector.

[0256] "Polynucleotide," or "nucleic acid," as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be separated by non-nucleotide components. A polynucleotide can include modification(s) generated after synthesis, such as conjugation to a label. Other types of modifications include, for example, "caps" that replace one or more of the naturally occurring nucleotides with an analog; internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.); those containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.); those with intercalating agents (e.g., acridine, psoralen, etc.); those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.); those containing alkylating agents; those with modified linkages (e.g., alpha anomeric nucleic acids, etc.); and unmodified forms of the polynucleotide(s). Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, with a phosphonate group, a phosphate group, protected with a standard protecting group, or activated to generate additional bonds to additional nucleotides, or conjugated to a solid or semi-solid support. The 5' and 3' terminal OH may be phosphorylated or substituted with an amine or an organic capping group moiety of 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.Polynucleotides may contain analog forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl, optionally containing an ether (-O-) linkage. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0257] A "host cell" includes an individual cell or cell culture that can be and has been a recipient for vector(s) for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, spontaneous, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of the present disclosure.

[0258] As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals exposed thereto at the dosages and concentrations employed. Often, physiologically acceptable carriers are aqueous pH buffers. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions, such as sodium; and / or nonionic surfactants, such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0259] The term "prophylaxis" is art-recognized and refers to the prevention or prophylaxis of a blood disorder (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antithrombotic agents). When used in connection with conditions such as phospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin)), it is well understood in the art and includes administration of a composition that reduces the frequency or severity of, or delays the onset of, one or more symptoms of the medical condition in a subject compared to a subject not ingesting the composition. Thus, blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS),Prevention of an autoimmune disorder (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), an infectious disease (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), an immune complex disease (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or a drug-induced hematological disorder (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin) includes, e.g., increasing platelet counts by a statistically and / or clinically significant amount in a population of patients receiving therapy compared to a control population not receiving therapy. Similarly, blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatible acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatible acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell Alloimmunization, Felty's syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus, leukemia, thrombocytopenia ...rombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus, leukemia, thrombocytopenia, thrombosis, thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS)), autoimmune disorders (e.g., systemic lupus erythematosus, leukemia, thrombocytopenia, thrombosis, thrombocytopenia, thrombosis, vasculitis, lupus nephritis, thrombocytopenia, thrombosis, thrombocytopenia, thrombosis, vasculitis, lupu Prevention of infectious diseases (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia resulting from drugs such as penicillin, quinine, or heparin) is recommended if the patient receiving therapy has a blood disorder (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome,ABO incompatible acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty's syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease) , ulcerative colitis), infections (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin) or related conditions, compared to patients not receiving the therapy.

[0260] The term "subject," as used herein, refers to a living mammal and may be used interchangeably with the term "patient." Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates, such as chimpanzees, and other ape and monkey species; farm animals, such as cows, horses, sheep, goats, and pigs; domestic animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs. The term does not denote a particular age or sex.

[0261] As used herein, the term "treating" or "treatment" includes reducing, arresting, or reversing the symptoms, clinical signs, or underlying pathology of a condition to stabilize or improve a subject's condition or make it less likely that the subject's condition will worsen to the same extent as if the subject had not received treatment.

[0262] The term "therapeutically effective amount" of a compound in the context of a subject treatment method refers to the amount of compound(s) in a preparation that, when administered as part of a desired dosing regimen (to a mammal, preferably a human), will treat a disease or condition, for example, to a clinically acceptable standard for cosmetic purposes, alleviate symptoms, ameliorate disease conditions, or delay the onset of a disease condition, at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutically effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in the individual.

[0263] As used herein, an individual "at risk" of developing a particular disease, disorder, or condition may or may not exhibit detectable disease or disease symptoms, and may or may not exhibit detectable disease or disease symptoms prior to the treatment methods described herein. "At risk" means that an individual has one or more risk factors, which are measurable parameters that correlate with the development of a particular disease, disorder, or condition, as known in the art. Individuals with one or more of these risk factors are more likely to develop a particular disease, disorder, or condition than individuals without one or more of these risk factors.

[0264] "Chronic" administration refers to the administration of a pharmaceutical agent(s) continuously, as opposed to acutely, so as to maintain the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration refers to treatment that is cyclical / periodic in nature, rather than administered continuously without interruption.

[0265] As used herein, administration "in conjunction with" another compound or composition includes simultaneous administration and / or administration at different times. Concurrent administration also encompasses administration as a co-formulation or as separate compositions, including using different dosing frequencies or intervals, and the same or different routes of administration.

[0266] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention 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 cited herein are incorporated herein by reference, and the methods and / or materials for which the publications are cited are not intended to be limiting unless specifically stated otherwise, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (F.M.A.usubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988); Antibodies, A Laboratory Manual, and Animal Cell Culture (R.I. Freshney, ed. (1987)); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(JECellis,ed.,1998)Academic Press;Animal Cell Culture(RIFreshney),ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMiller and MP Calos, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press,1988-1989);Monoclonal Antibodies:A Practical Approach(P.Shepherd and The present invention discloses and describes widely used methodologies described in "Using Antibodies: A Laboratory Manual" (E. Harlow and D. Lane, eds., Oxford University Press, 2000); "The Antibodies" (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and "Cancer: Principles and Practice of Oncology" (VT DeVita et al., eds., J.B. Lippincott Company, 1993).

[0267] Nucleic acids, vectors and host cells Antibodies suitable for use in the methods of the present disclosure can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In some embodiments, isolated nucleic acids having a nucleotide sequence encoding any of the antibodies of the present disclosure are provided. Such nucleic acids may be used in combination with the V of an anti-C1q, anti-C1r, or anti-C1s antibody. L / C L and / or V H / C H In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. Host cells containing such nucleic acids may also be provided. The host cells may encode an amino acid sequence containing (1) the V of the antibody. L / C L The amino acid sequence containing the V H / C H (2) a vector containing a nucleic acid encoding an amino acid sequence containing 1, or (3) a V L / C L a first vector containing a nucleic acid encoding an amino acid sequence containing H / C H The host cell may contain (e.g., be transduced with) a second vector containing a nucleic acid encoding an amino acid sequence containing 1. In some embodiments, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In some embodiments, the host cell is a bacterium, such as E. coli.

[0268] Disclosed herein are methods for producing anti-C1q, anti-C1r, or anti-C1s antibodies. The methods include culturing a host cell of the disclosure containing nucleic acid encoding an anti-C1q, anti-C1r, or anti-C1s antibody under conditions suitable for expression of the antibody. In some embodiments, the antibody is then recovered from the host cell (or host cell culture medium).

[0269] For recombinant production of a humanized anti-C1q, anti-C1r, or anti-C1s antibody of the present disclosure, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains).

[0270] In certain embodiments, the present disclosure provides anti-C1q antibody Fab fragments, anti-C1s antibody Fab fragments, and anti-C1r antibody Fab fragments that bind to C1q, C1s, and C1r proteins, respectively. The high-affinity Fab fragments of these antibodies are suitable for selectively inhibiting complement activation in the blood space. The high-affinity Fab fragments of these antibodies are suitable for administration, for example, subcutaneous, intramuscular, and intravascular administration.

[0271] Suitable vectors containing a nucleic acid sequence encoding an antibody of the present disclosure, or any of the fragment polypeptides thereof (including antibodies) described herein, include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques or selected from a large number of cloning vectors available in the art. While the cloning vector selected can vary depending on the host cell intended for use, useful cloning vectors will typically be capable of autonomous replication, possess a single target for a particular restriction endonuclease, and / or contain a gene for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, e.g., pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Stratagene, and Invitrogen.

[0272] A vector containing a nucleic acid of interest can be introduced into a host cell by any of a number of suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; particle bombardment; lipofection; and infection (e.g., the vector is an infectious agent such as vaccinia virus). The choice of introducing a vector or polynucleotide often depends on the characteristics of the host cell. In some embodiments, the vector contains a nucleic acid containing one or more amino acid sequences encoding an anti-C1q, anti-C1r, or anti-C1s antibody of the present disclosure.

[0273] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, the anti-C1q, anti-C1r, or anti-C1s antibodies of the present disclosure can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523; and Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254 (describing the expression of antibody fragments in E. coli)). In other embodiments, antibodies of the present disclosure can be produced in eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells) (e.g., U.S. Patent Application No. 14 / 269,950, U.S. Patent No. 8,981,071, Eur J Biochem. 1991 Jan 1;195(1):235-42). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0274] Antibody screening Candidate antibodies can be screened for their ability to modulate complement activation. Such screening can be performed using in vitro models, genetically altered cells or animals, or purified proteins. A variety of assays, such as in vitro culture systems, can be used for this purpose.

[0275] Candidate antibodies can also be identified using computer-based modeling, binding assays, etc. Various in vitro models can be used to determine whether an antibody binds or otherwise affects complement activity. Such candidate antibodies can be tested by contacting them with plasma from healthy donors and determining complement activation (e.g., by antigen C3c capture ELISA). Such antibodies may be used to treat blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura ( In vivo models may be further tested for efficacy against ITP, thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin).

[0276] Typically, multiple assays are run in parallel at different antibody concentrations to obtain differential responses to various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.

[0277] Pharmaceutical Compositions and Administration The complement inhibitors (eg, antibodies) of the present disclosure can be administered in the form of a pharmaceutical composition.

[0278] Therapeutic formulations of the inhibitors (e.g., antibodies, antibody fragments, and / or antibody derivatives) of the present disclosure may be prepared in the form of a lyophilized formulation or aqueous solution for storage by mixing the inhibitor having the desired purity with any pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.

[1980] ). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers, e.g., phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., blood Examples of suitable surfactants include serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS™, or polyethylene glycol (PEG).

[0279] Lipofections or liposomes can also be used to deliver the antibody or antibody fragment, or antibody derivative, into cells, preferably an epitope or minimal fragment that specifically binds to the binding domain of the target protein.

[0280] The inhibitors may also be incorporated into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0281] The formulations to be used for administration can be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0282] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the inhibitor, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time.

[0283] The antibodies, antibody fragments, and / or antibody derivatives and compositions of the disclosure are typically administered by a variety of routes, including, but not limited to, topical, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and intralesional administration. Parenteral routes of administration include intramuscular, intravenous, intraarterial, intraperitoneal, intrathecal, or subcutaneous administration.

[0284] Pharmaceutical compositions may also contain pharmaceutically acceptable, non-toxic diluents, defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration, depending on the desired formulation. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. Pharmaceutical compositions or formulations may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, etc. The compositions may also contain additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents.

[0285] The composition may also include any of a variety of stabilizers, such as, for example, antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide may be complexed with a variety of well-known compounds that enhance the polypeptide's in vivo stability or otherwise improve its pharmacological properties (e.g., increase the polypeptide's half-life, reduce its toxicity, improve other pharmacokinetic and / or pharmacodynamic properties, or improve solubility or uptake). Examples of such modifying or complexing agents include sulfate, gluconate, citrate, and phosphate. The polypeptides of the composition may also be complexed with molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids. Further guidance regarding formulations suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990).

[0286] Toxicity and therapeutic efficacy of an active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, including, for example, determining LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit a high therapeutic index are preferred.

[0287] Data obtained from cell culture and / or animal studies and / or human clinical trials can be used to formulate a range of dosages for humans. The dosage of the active ingredient typically falls within a range of circulating concentrations that include the ED50 with low toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized.

[0288] The pharmaceutical compositions described herein can be administered in a variety of different ways, including administering the composition containing a pharmaceutically acceptable carrier via oral, intranasal, rectal, topical, intraperitoneal, intravenous, intramuscular, subcutaneous, subdermal, transdermal, intrathecal, and intracranial methods.

[0289] Formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives.

[0290] The components used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food grade, usually at least analytical grade, and more typically at least pharmaceutical grade). Furthermore, compositions intended for parenteral use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic substances, particularly any endotoxins, that may be present during the synthesis or purification process. Compositions for parenteral administration are also typically substantially isotonic and produced under GMP conditions.

[0291] The compositions of the present disclosure can be administered using any medically appropriate procedure, for example, intravascular (intravenous, intraarterial, intracapillary), intramuscular, or subcutaneous administration. The compositions can be administered via an autoinjector or infusion device, for example, a minipump or on-body infusion device.

[0292] The effective amount of a therapeutic composition given to a particular patient may depend on a variety of factors, some of which may vary from patient to patient. A competent clinician can determine the effective amount of a therapeutic agent to administer to a patient. The dosage of the agent will depend on the treatment, the route of administration, the characteristics of the therapeutic agent, the patient's sensitivity to the therapeutic agent, etc. Using LD50 animal data and other information, a clinician can determine the maximum safe dose for an individual depending on the route of administration. Using conventional techniques, a competent clinician can optimize the dosage of a particular therapeutic composition during routine clinical trials. The composition may be administered to a subject in a series of multiple doses. For therapeutic compositions, regular periodic administration may sometimes be necessary or desirable. Treatment regimens vary depending on the agent; for example, some agents may be taken long-term on a once-daily or twice-daily basis, while more selective agents may be administered once-daily, twice-daily, twice-weekly, once-weekly, etc., for a more defined period, such as one, two, three, or more days, one week or more, one month or more, etc.

[0293] In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of between 10 mg / kg and 150 mg / kg. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of between 10 mg / kg and 20 mg / kg, 20 mg / kg and 30 mg / kg, 30 mg / kg and 40 mg / kg, 40 mg / kg and 50 mg / kg, 50 mg / kg and 60 mg / kg, 60 mg / kg and 70 mg / kg, 70 mg / kg and 80 mg / kg, 80 mg / kg and 90 mg / kg, 90 mg / kg and 100 mg / kg, 100 mg / kg and 110 mg / kg, 110 mg / kg and 120 mg / kg, 120 mg / kg and 130 mg / kg, 130 mg / kg and 140 mg / kg, or 140 mg / kg and 150 mg / kg. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of between 75 mg / kg and 100 mg / kg. The antibody may be administered weekly, every two weeks, or monthly. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 75 mg / kg. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 100 mg / kg. The antibody may be administered weekly, every two weeks, every three weeks, or monthly. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 75 mg / kg once weekly. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 75 mg / kg once every two weeks. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 75 mg / kg once every three weeks. In some embodiments, the antibody is administered to a subject monthly by intravenous injection or infusion at a dose of 75 mg / kg. In some embodiments, the antibody is administered to a subject weekly by intravenous injection or infusion at a dose of 100 mg / kg. In some embodiments, the antibody is administered to a subject every two weeks by intravenous injection or infusion at a dose of 100 mg / kg. In some embodiments, the antibody is administered to a subject every three weeks by intravenous injection or infusion at a dose of 100 mg / kg.In some embodiments, the antibody is administered to the subject monthly by intravenous injection or infusion at a dose of 100 mg / kg. In some embodiments, the antibody is administered to the subject by subcutaneous or intramuscular injection at a dose between 1 mg / kg and 10 mg / kg. In some embodiments, the antibody is administered to the subject by subcutaneous or intramuscular injection at a dose between 1 mg / kg and 3 mg / kg, 3 mg / kg and 5 mg / kg, 5 mg / kg and 7 mg / kg, or 7 mg / kg and 10 mg / kg. In some embodiments, the antibody is administered daily, every other day, weekly, biweekly, or monthly.

[0294] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is administered to a subject by intravenous injection or infusion, intramuscular injection, or subcutaneous injection. In some embodiments, the antibody fragment is administered at a dose between 0.1 mg / kg and 50 mg / kg. In some embodiments, the antibody fragment is administered at a dose between 0.1 mg / kg and 1 mg / kg, 1 mg / kg and 5 mg / kg, 5 mg / kg and 10 mg / kg, 10 mg / kg and 15 mg / kg, 15 mg / kg and 20 mg / kg, 20 mg / kg and 25 mg / kg, 25 mg / kg and 30 mg / kg, 30 mg / kg and 35 mg / kg, 35 mg / kg and 40 mg / kg, 40 mg / kg and 45 mg / kg, or 45 mg / kg and 50 mg / kg. In some embodiments, the antibody fragment is administered at a dose between 0.3 mg / kg and 10 mg / kg. In some embodiments, the antibody fragment is administered daily, every other day, weekly, biweekly, or monthly. In some embodiments, the antibody fragment is administered at an initial, pre-dosing dose that is higher than the daily, biweekly, weekly, biweekly, or monthly dose. In some embodiments, the initial, pre-dosing dose is between 3 mg / kg and 50 mg / kg. In some embodiments, the initial, pre-dosing dose is between 3 mg / kg and 5 mg / kg, 5 mg / kg and 10 mg / kg, 10 mg / kg and 15 mg / kg, 15 mg / kg and 20 mg / kg, 20 mg / kg and 25 mg / kg, 25 mg / kg and 30 mg / kg, 30 mg / kg and 35 mg / kg, 35 mg / kg and 40 mg / kg, 40 mg / kg and 45 mg / kg, or 45 mg / kg and 50 mg / kg. In some embodiments, the initial, pre-dosing dose is between 3 mg / kg and 20 mg / kg. In some embodiments, the antibody fragment has a shorter half-life compared to its corresponding full-length antibody, e.g., the antibody fragment is rapidly cleared, thereby sparing C1q activity outside the blood space of the subject, or the antibody selectively inhibits C1q within the blood space of the subject, thereby sparing C1q activity outside the blood space of the subject. In some embodiments, the blood space is limited within a blood vessel, such as an artery, arteriole, capillary, venule, or vein. The blood space may contain serum, platelets, endothelial cells, blood cells, or hematopoietic cells.In some embodiments, inhibiting C1q in the blood space of a subject reduces tissue damage in highly vascularized tissues. Examples of highly vascularized tissues are the kidney, alveoli, capillary beds, or glomeruli.

[0295] Formulations can be optimized for retention and stabilization within the body, including within the blood space. In some embodiments, when an agent is administered to the blood space, it is desirable that the agent be retained within the blood space and not diffuse or otherwise distribute extravascularly (e.g., into surrounding tissues). Stabilization techniques include cross-linking, multimerization, or linking to groups such as polyethylene glycol, polyacrylamide, neutral protein carriers, etc., to achieve increased molecular weight.

[0296] Other strategies for increasing retention include incorporating the drug in a biodegradable or bioerodible implant. The rate of release of the therapeutically active agent is controlled by the rate of transport through the polymeric matrix and the biodegradation of the implant. Drug transport through a polymer barrier is also influenced by compound solubility, polymer hydrophilicity, the degree of polymer crosslinking, the swelling of the polymer upon water absorption so that the polymer barrier becomes more permeable to the drug, and the shape of the implant. The implant is dimensioned to suit the size and shape of the area selected as the site of implantation. The implant can be a particle, sheet, patch, plaque, fiber, microcapsule, etc., and can be of any size or shape that is compatible with the selected insertion site.

[0297] Implants can be monolithic (i.e., have the active agent uniformly distributed throughout the polymer matrix) or encapsulated (a reservoir of active agent is encapsulated by the polymer matrix). The choice of polymer composition used will depend on the site of administration, desired duration of treatment, patient tolerance, the nature of the disease being treated, etc. Polymer characteristics include biodegradability at the site of implantation, compatibility with the drug of interest, ease of encapsulation, and half-life in a physiological environment.

[0298] Biodegradable polymer compositions that can be used include organic esters or ethers that, upon degradation, yield physiologically acceptable degradation products containing the monomer. Anhydrides, amides, orthoesters, and the like can be utilized, either by themselves or in combination with other monomers. The polymers can be condensation polymers. The polymers can be crosslinked or non-crosslinked. Of particular interest are polymers of hydroxyaliphatic carboxylic acids (either homopolymers or copolymers) and polysaccharides. Polyesters of interest include polymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and combinations thereof. By using L-lactic acid or D-lactic acid, gradually biodegrading polymers can be achieved, while degradation is substantially accelerated with the racemate. Copolymers of glycolic acid and lactic acid are of particular interest, in which the rate of biodegradation is controlled by the ratio of glycolic acid to lactic acid. The most rapidly degrading copolymers have roughly equal amounts of glycolic acid and lactic acid, with either homopolymer being more resistant to degradation. The ratio of glycolic acid to lactic acid also affects the brittleness of the implant, with more flexible implants being desirable for larger geometries. Polysaccharides of interest include calcium alginate and functionalized celluloses, particularly carboxymethylcellulose esters, which are water-insoluble and characterized by a molecular weight of approximately 5 kD to 500 kD. Biodegradable hydrogels may also be used in the implants of the present disclosure. Hydrogels are typically copolymeric materials characterized by their ability to absorb fluid. Exemplary biodegradable hydrogels that may be used are described in Heller in: Hydrogels in Medicine and Pharmacy, NAPeppes ed., Vol. III, CRC Press, Boca Raton, Fla., 1987, pp. 137-149.

[0299] kit The present disclosure also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition. Associated with such container(s) may be a notice in a form prescribed by a governmental authority regulating the manufacture, use, or sale of pharmaceutical or biological products, the notice reflecting approval by the authority of the manufacture, use, or sale for human administration.

[0300] Kits of the present disclosure can include one or more containers containing purified anti-C1q, anti-C1r, or anti-C1s antibodies and instructions for use according to methods known in the art. Typically, these instructions include instructions for administering the inhibitor to treat or diagnose a disease according to any method known in the art. The kit may be used to monitor the progression of a blood disorder (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis ... The method may further include instructions for selecting an individual suitable for treatment based on identification of whether or not the individual has an autoimmune disorder (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), an infectious disease (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), an immune complex disease (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or a drug-induced hematological disorder (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin).

[0301] The instructions typically include information regarding dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. The instructions supplied in the kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0302] The label or package insert may indicate that the composition is intended to treat a blood disorder (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura, The method may be indicated for use in treating an autoimmune disorder (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), an infectious disease (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), an immune complex disease (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or a drug-induced hematological disorder (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin). Instructions may be provided for practicing any of the methods described herein.

[0303] The kit of the present disclosure is preferably placed in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Packages for use in combination with specific devices, such as inhalers, nasal administration devices (e.g., nebulizers), auto-injectors, or injection devices, such as mini-pumps or on-body injectors, are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an inhibitor of the classical complement pathway. The container may further contain a second pharmaceutically active agent.

[0304] Kits may optionally provide additional components, such as buffers and instructional information. Typically, the kit includes a container and a label or package insert(s) on or associated with the container.

[0305] Target pathology Representative conditions of interest include a variety of blood disorders and other hematological diseases.

[0306] The term "blood disorder" or "hematological disease" is used in the broadest sense and includes any pathological condition involving acute or chronic blood pathologies. Such diseases are typically characterized by thrombosis, inflammation, and hemolysis.

[0307] A variety of hematological conditions are targeted for this method of preventing, reducing the risk of developing, or treating hematological disorders, which involves administering antibodies, antibody fragments, and / or antibody derivatives that bind to complement components C1q, C1r, or C1s. Such conditions include cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatible acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatible acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITH), and thrombocytopenic purpura (THP). ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin).

[0308] Autoimmune hemolytic anemia (AIHA), also known as "immune hemolytic anemia," occurs when antibodies directed against a subject's own red blood cells (RBCs) rupture (lyse) them, resulting in insufficient plasma concentrations. RBC lifespan is reduced from a normal 100-120 days to only a few days in severe cases. RBC intracellular components are released into the circulation and tissues, resulting in some of the characteristic symptoms of this condition. Antibodies are usually directed against high-frequency antigens and commonly act on allogeneic RBCs (RBCs derived from a non-self source, e.g., in the case of blood transfusions). AIHA is classified as either warm or cold autoimmune hemolytic anemia (including cold agglutinin disease and paroxysmal cold hemoglobinuria). These classifications are based on the characteristics of the autoantibodies involved in the pathogenesis of the disease. Each has different underlying causes, management, and prognosis, making classification important when treating patients with AIHA.

[0309] Cold agglutinin disease is a type of autoimmune hemolytic anemia in which the body's immune system mistakenly attacks and destroys its own red blood cells. When an affected person's blood is exposed to low temperatures (32° to 50°F), certain proteins (IgM antibodies) that normally attack bacteria attach themselves to red blood cells, causing them to bind to each other and clump together (agglutination). This ultimately leads to the premature destruction of red blood cells (hemolysis), resulting in anemia and other related signs and symptoms. Cold agglutinin disease can be primary (of unknown cause) or secondary to an underlying condition such as an infection, another autoimmune disease, or certain cancers. Treatment depends on many factors, including the severity of the condition, the signs and symptoms present in each individual, and the underlying cause.

[0310] Symptoms include, for example, pain, fever, pallor, jaundice, hives (skin rash), hemoglobinuria, hemoglobinemia, anemia, and kidney disease or acute renal failure. Symptoms can occur after exposure to cold temperatures.

[0311] A subject can be identified as having CAD using an assay to detect the presence or amount (titer) of agglutinating autoantibodies that bind to the "I antigen" on red blood cells. The antibodies can be monoclonal (e.g., monoclonal IgM or IgA) or polyclonal. A subject can also be diagnosed as having CAD using one or more of a complete blood count (CBC), urinalysis, biochemical studies, and a Coombs test to test for hemolysis in the blood. For example, biochemical studies can be used to detect elevated lactase dehydrogenase levels, elevated unconjugated bilirubin levels, low haptoglobin levels, and / or the presence of free plasma hemoglobin, all of which can be indicative of acute hemolysis. Other tests that can be used to detect CAD include detecting complement levels in serum. For example, measured plasma complement levels (e.g., C2, C3, and C4) are decreased in CAD due to consumption during the acute phase of hemolysis.

[0312] Warm agglutinin hemolytic anemia is an autoimmune disorder characterized by the premature destruction of healthy red blood cells by autoantibodies. In most cases, the cause of warm antibody hemolytic anemia is unknown. These cases may be referred to as primary warm antibody hemolytic anemia or idiopathic warm antibody hemolytic anemia. The disorder may also occur as part of a larger disorder. Such cases are known as secondary warm antibody hemolytic anemia. The specific symptoms that arise may vary and depend on the rate of onset, the rate of destruction of healthy red blood cells, and the presence of an underlying disorder. Some individuals, particularly those with a gradual onset of anemia, may not have any obvious symptoms (asymptomatic). Affected individuals may eventually develop abnormal paleness of the skin (pallor), fatigue, dyspnea on exertion, dizziness, and palpitations. Yellowing of the skin and whites of the eyes (jaundice) and an enlarged spleen (splenomegaly) are also common findings in individuals with warm antibody hemolytic anemia. Splenomegaly can cause affected individuals to experience a feeling of abdominal fullness or fullness. Occasionally, an enlarged liver (hepatomegaly) can also occur in some cases. Individuals with severe cases, particularly those with rapid (acute) onset, can develop more serious complications, including loss of consciousness (fainting), chest pain (angina), abnormally rapid heartbeat (tachycardia), and heart failure. Some individuals have a rare form of warm antibody hemolytic anemia caused by IgM antibodies (as opposed to the more common form caused by IgG antibodies).

[0313] Autoimmune thrombocytopenia (ITP) is usually known as isolated platelet count thrombocytopenia in patients with a normal bone marrow and no other cause of thrombocytopenia. It causes a characteristic purpuric rash and an increased tendency to bleed. Two distinct clinical syndromes manifest as an acute form in children and a chronic form in adults. The acute form often occurs after an infection and resolves spontaneously within 2 months. Chronic immune thrombocytopenia persists for longer than 6 months, and the specific cause is unknown.

[0314] ITP is diagnosed by a low platelet count on a complete blood count (a common blood test). However, because the diagnosis depends on excluding other causes of low platelet counts, additional investigations, such as a bone marrow biopsy, may be necessary in some cases.

[0315] In mild cases, only careful observation may be required, but extremely low counts or significant bleeding may prompt treatment with corticosteroids, intravenous immunoglobulin, anti-D immunoglobulin, or immunosuppressants. Refractory ITP (not responsive to conventional treatments) may require splenectomy. Severe bleeding with extremely low counts may require platelet transfusions. Sometimes the body compensates by producing abnormally large platelets.

[0316] Symptoms include spontaneous bruising (purpura) and petechiae (small bruises), especially on the extremities, bleeding from the nostrils and / or gums, and menorrhagia (excessive menstrual bleeding), any of which may occur when the platelet count is below 20,000 per μL. Extremely low counts (<10,000 per μL) can result in spontaneous hematomas (blood clots) in the mouth or on other mucous membranes. Bleeding times from minor lacerations or abrasions are usually prolonged. Severe and potentially fatal complications of extremely low counts (<5,000 per μL) include subarachnoid or intracerebral hemorrhage (bleeding in the skull or brain), lower gastrointestinal bleeding, or other internal bleeding. ITP patients with extremely low counts are prone to internal bleeding caused by blunt abdominal trauma, such as may be experienced in a car accident. These complications are less likely when the platelet count exceeds 20,000 per μL.

[0317] Antiphospholipid syndrome (APS), also known as Hughes syndrome, is an autoimmune hypercoagulable state commonly caused by antiphospholipid antibodies. APS can lead to blood clots in arteries and veins (thrombosis) and pregnancy-related complications, such as miscarriage, stillbirth, premature birth, and severe preeclampsia.

[0318] Diagnostic criteria require one clinical event, i.e., thrombosis or pregnancy complications, and two antibody blood tests, typically at least 3 months apart, confirming the presence of either lupus anticoagulant or anti-β2-glycoprotein-I; because β2-glycoprotein-I antibodies are a subset of anticardiolipin antibodies, anticardiolipin assays can be performed as a less specific surrogate.

[0319] Antiphospholipid syndrome can be primary or secondary. Primary antiphospholipid syndrome occurs in the absence of any other associated diseases. Secondary antiphospholipid syndrome occurs with other autoimmune diseases, such as systemic lupus erythematosus (SLE). In rare cases, APS leads to rapid organ failure due to systemic thrombosis; this is called "fulminant antiphospholipid syndrome" (CAPS) and is associated with a high risk of death. Antiphospholipid syndrome often requires treatment with anticoagulants, such as heparin, to reduce the risk of further thrombosis and improve pregnancy outcomes.

[0320] Evans syndrome is a chronic hematological disorder typically characterized by the simultaneous or sequential association of autoimmune hemolytic anemia and immune thrombocytopenic purpura (ITP). The syndrome can manifest in both childhood and adulthood. The onset of thrombocytopenia may precede, occur simultaneously with, or follow the onset of AIHA. The severity of symptoms and the delay between the onset of AIHA and / or ITP vary. In non-synchronous adult cases, the delay between onset averages 4 years. ITP is often manifested by mucocutaneous bleeding with epistaxis, petechiae, purpura, and ecchymoses. In cases of severe thrombocytopenia, hematuria, gastrointestinal bleeding, and / or meningeal hemorrhage may be observed in rare cases.

[0321] Evans syndrome is an autoimmune disorder in which non-cross-reactive autoantibodies are targeted against different antigenic determinants on red blood cells, platelets, and occasionally neutrophils; however, the exact pathophysiological mechanism is unknown. The observation of a decrease in T-helper and an increase in T-suppressor lymphocyte populations suggests that the cytopenia may be related to a T-cell abnormality. Evans syndrome is often associated with other diseases, such as systemic lupus erythematosus, antiphospholipid syndrome, autoimmune lymphoproliferative syndrome, and common variable immunodeficiency.

[0322] Diagnosis is based on a complete blood count demonstrating anemia (hemoglobin level <12 g / dL) and thrombocytopenia (platelet count <100,000 / microL), with or without associated neutropenia (neutrophil count <1500 / microL). Elevated lactate dehydrogenase (LDH) and / or direct bilirubin levels and decreased haptoglobin levels may indicate hemolysis. A positive direct antiglobulin test (Coombs' test) confirms the presence of antibodies targeting red blood cell (RBC) antigens. The presence of autoantibodies targeting both platelets and neutrophils may also be observed.

[0323] The differential diagnosis primarily includes microangiopathy (e.g., thrombotic or thrombocytopenic purpura). Most cases are sporadic. Familial cases have been observed exceptionally, primarily in the setting of an underlying primary immunodeficiency.

[0324] Immunosuppressive therapy, often combined with intravenous immunoglobulin for ITP, constitutes first-line treatment. Administration of corticosteroids (prednisone) is the mainstay of treatment, but other drugs such as rituximab, cyclosporine, azathioprine, cyclophosphamide, and danazol may be prescribed for refractory cases. Splenectomy is performed as a third-line treatment; however, long-term remissions are infrequent, and patients are at high risk for sepsis. In severe cases, hematopoietic stem cell transplantation may be required. Regardless of treatment, Evans syndrome can have alternating periods of remission and relapse of AIHA and / or ITP, which can be associated with significant morbidity and mortality due to severe bleeding and infection in cases of severe thrombocytopenia and neutropenia.

[0325] Neonatal alloimmune thrombocytopenia (NAIT), also known as fetal and neonatal alloimmune thrombocytopenia (FNAIT), is a blood disorder affecting fetuses and newborns, resulting in a low platelet count (thrombocytopenia). Platelet antigens are inherited from both the father and mother. FNAIT is typically caused by antibodies specific to paternally inherited platelet antigens that are not present in the mother. Through fetal-maternal transfusion (or fetal-maternal hemorrhage), these antigens become recognized as non-self by the mother's immune system, followed by the generation of alloreactive antibodies that cross the placenta. NAIT is usually caused by the transplacental transfer of maternal platelet-specific alloantibodies and, rarely, human leukocyte antigen (HLA) alloantibodies (expressed by platelets) to the fetus, whose platelets express the corresponding antigens.

[0326] Thrombocytopenia is usually mild, and affected newborns remain largely asymptomatic. In these cases, therapeutic intervention is not indicated. In severe thrombocytopenia, newborns may present with bleeding complications at birth or within hours of birth. The most severe complication is intracranial hemorrhage, which results in death in approximately 10% of cases and neurological sequelae in 20%.

[0327] Approximately 80% of NAIT cases are caused by antibodies against the platelet antigen HPA-1a, 15% by anti-HPA-5b, and 5% by other antibodies (e.g., HPA-1b, HPA-15, HPA-3, and HPA-9b). HPA-1a is present in 98% of the US population, suggesting that approximately 2% of women who are HPA-1a negative may be at risk for FNAIT during pregnancy.

[0328] Unlike hemolytic disease of the fetus and newborn (HDFN), NAIT occurs during the first trimester in up to 50% of cases. Affected fetuses can develop severe thrombocytopenia (<50,000 / μL) very early in pregnancy (as early as 20 weeks, coinciding with the development of platelet antigens and the majority of time in utero). Thrombocytopenia usually increases as pregnancy progresses. During the first trimester, NAIT is often not detected until birth, when the newborn exhibits typical symptoms of thrombocytopenia, including petechiae, bruising, or intracranial hemorrhage. Intrauterine intracranial hemorrhage occurs in approximately 10%–30% of affected cases. NAIT is thought to be the underlying cause in the majority of cases of intracranial hemorrhage due to thrombocytopenia. The risk of bleeding is inversely correlated with the platelet count, with the highest risk occurring when the platelet count is below 100,000 / μL.

[0329] Recurrence of NAIT is estimated to be greater than 80% in subsequent pregnancies with incompatible fetuses (i.e., subsequent pregnancies that also carry the target platelet antigen). Subsequent cases of NAIT may be of similar or greater severity. The fetal response to FNAIT varies and may include compensatory extramedullary hematopoiesis. Rarely, hydrops fetalis may develop. Fetal anemia (in the absence of red blood cell incompatibility) may also occur.

[0330] Treatment method By administering the agent that inhibits complement activation, complement deposition on blood cells is prevented.Such agents include anti-C1q, anti-C1r or anti-C1s antibody inhibitors.Other agents can include the inhibitor that upregulates the expression of natural complement, or the agent that downregulates the synthesis of C1q, C1r or C1s in platelets or blood cells (e.g., erythrocytes, monocytes, neutrophils), the agent that blocks complement activation, the agent that blocks the signal for complement activation, etc.

[0331] In some aspects, methods for preventing, reducing the risk of developing, or treating a blood disorder are disclosed. Such methods include administering a C1q inhibitor to a subject. Numerous embodiments are further provided that may be applied to any aspect of the invention described herein. For example, in some embodiments, the C1q inhibitor is an antibody, an aptamer, an antisense nucleic acid, or a gene editing agent. In some embodiments, the inhibitor is an anti-C1q antibody. The anti-C1q antibody may inhibit the interaction between C1q and autoantibodies, or between C1q and C1r, or between C1q and C1s, or may promote the clearance of C1q from the circulation or tissues. In some embodiments, the anti-C1q antibody has a dissociation constant (K) in the range of 100 nM to 0.005 nM or less than 0.005 nM. D In some embodiments, the anti-C1q antibody binds to C1q with a binding stoichiometry ranging from 20:1 to 1.0:1 or less than 1.0:1, a binding stoichiometry ranging from 6:1 to 1.0:1 or less than 1.0:1, or a binding stoichiometry ranging from 2.5:1 to 1.0:1 or less than 1.0:1.

[0332] The method inhibits the biological activity of C1q, C1r, or C1s, for example, (1) C1q binding to autoantibodies, (2) C1q binding to C1r, (3) C1q binding to C1s, (4) C1q binding to phosphatidylserine, (5) C1q binding to pentraxin-3, (6) C1q binding to C-reactive protein (CRP), (7) C1q binding to globular C1q receptor (gC1qR), (8) C1q binding to complement receptor 1 (CR1), (9) C1q binding to B-amyloid, or (10) C1q binding to calreticulin.In other embodiments, the biological activity of C1q is (1) activation of the classical complement pathway, (2) reduced lysis and / or reduced C3 deposition, (3) activation of antibody and complement dependent cytotoxicity, (4) CH50 hemolysis, (5) reduced erythrocyte lysis, (6) reduced erythrophagocytosis, (7) reduced dendritic cell infiltration, (8) inhibition of complement mediated erythrocyte lysis, (9) reduced lymphocyte infiltration, (10) reduced macrophage infiltration, (11) reduced antibody deposition, (12) Decreased neutrophil infiltration, (13) decreased platelet phagocytosis, (14) decreased platelet lysis, (15) improved graft survival, (16) decreased macrophage-mediated phagocytosis, (17) decreased autoantibody-mediated complement activation, (18) decreased red blood cell destruction due to transfusion reactions, (19) decreased alloantibody-mediated red blood cell lysis, (20) decreased hemolysis due to transfusion reactions, (21) decreased alloantibody-mediated platelet lysis, (22) improved anemia, (23) decreased eosinophilia, (24 ) Decreased C3 deposition on red blood cells (e.g., decreased C3b, iC3b, etc. deposition on RBCs), (25) Decreased C3 deposition on platelets (e.g., decreased C3b, iC3b, etc. deposition on platelets), (26) Decreased anaphylatoxin production, (27) Decreased autoantibody-mediated rash formation, (28) Decreased autoantibody-induced lupus erythematosus, (29) Decreased red blood cell destruction in transfusion reactions, (30) Decreased platelet lysis in transfusion reactions, (31) Obesity These include decreased cell activation, (32) decreased mast cell histamine release, (33) decreased vascular permeability, (34) decreased complement deposition on graft endothelium, (35) B cell antibody production, (36) dendritic cell maturation, (37) T cell proliferation, (38) cytokine production, (39) microglial activation, (40) Arthus reaction, (41) decreased anaphylatoxin production in graft endothelium, or (42) activation of complement receptor 3 (CR3 / C3)-expressing cells.

[0333] In some embodiments, the CH50 hemolysis comprises human CH50 hemolysis. The antibody may be capable of neutralizing at least about 50% to about 100% of human CH50 hemolysis. The antibody may be capable of neutralizing about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of human CH50 hemolysis. The antibody may be capable of neutralizing at least 50% of CH50 hemolysis at a dose of less than 150 ng / ml, less than 100 ng / ml, less than 50 ng / ml, or less than 20 ng / ml.

[0334] In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a human antibody, a chimeric antibody, a monovalent antibody, a multispecific antibody, or an antibody fragment, or an antibody derivative thereof. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment, such as a Fab fragment. Examples of antibody fragments are Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, and single-chain antibody molecules. In some embodiments, the antibody comprises a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO:5, HVR-L2 having the amino acid sequence of SEQ ID NO:6, and HVR-L3 having the amino acid sequence of SEQ ID NO:7. In some embodiments, the antibody comprises a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO:9, HVR-H2 having the amino acid sequence of SEQ ID NO:10, and HVR-H3 having the amino acid sequence of SEQ ID NO:11. In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence at least about 95% identical to an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, wherein the light chain variable domain comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the light chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 4 and 35-38. In some embodiments, the antibody comprises a heavy chain variable domain comprising an amino acid sequence at least about 95% identical to an amino acid sequence selected from SEQ ID NOs: 8 and 31-34, wherein the heavy chain variable domain comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO: 11. In some embodiments, the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 8 and 31-34. In some embodiments, the antibody is an antibody fragment comprising a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40. The antibody may be administered by parenteral injection or infusion, such as subcutaneous or intramuscular injection, or intravenous injection or infusion.

[0335] In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of between 10 mg / kg and 150 mg / kg. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of between 10 mg / kg and 20 mg / kg, 20 mg / kg and 30 mg / kg, 30 mg / kg and 40 mg / kg, 40 mg / kg and 50 mg / kg, 50 mg / kg and 60 mg / kg, 60 mg / kg and 70 mg / kg, 70 mg / kg and 80 mg / kg, 80 mg / kg and 90 mg / kg, 90 mg / kg and 100 mg / kg, 100 mg / kg and 110 mg / kg, 110 mg / kg and 120 mg / kg, 120 mg / kg and 130 mg / kg, 130 mg / kg and 140 mg / kg, or 140 mg / kg and 150 mg / kg. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose between 75 mg / kg and 100 mg / kg. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 75 mg / kg. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 100 mg / kg. The antibody may be administered weekly, once every two weeks, once every three weeks, or once monthly. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 75 mg / kg once weekly. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 75 mg / kg once every two weeks. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 75 mg / kg once every three weeks. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 75 mg / kg once every three weeks. In some embodiments, the antibody is administered to the subject by intravenous injection or infusion at a dose of 75 mg / kg once monthly. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 100 mg / kg once a week. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 100 mg / kg every two weeks. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 100 mg / kg once every three weeks. In some embodiments, the antibody is administered to a subject by intravenous injection or infusion at a dose of 100 mg / kg once a month.The antibody may be administered weekly, every two weeks, or monthly. In some embodiments, the antibody is administered to a subject by subcutaneous or intramuscular injection at a dose of between 1 mg / kg and 10 mg / kg. In some embodiments, the antibody is administered to a subject by subcutaneous or intramuscular injection at a dose of between 1 mg / kg and 3 mg / kg, 3 mg / kg and 5 mg / kg, 5 mg / kg and 7 mg / kg, or 7 mg / kg and 10 mg / kg. In some embodiments, the antibody is administered daily, every other day, weekly, every two weeks, or monthly.

[0336] In some embodiments, the antibody is an antibody fragment. In some embodiments, the antibody fragment is administered to a subject by intravenous injection or infusion, intramuscular injection, or subcutaneous injection. In some embodiments, the antibody fragment is administered at a dose between 0.1 mg / kg and 50 mg / kg. In some embodiments, the antibody fragment is administered at a dose between 0.1 mg / kg and 1 mg / kg, 1 mg / kg and 5 mg / kg, 5 mg / kg and 10 mg / kg, 10 mg / kg and 15 mg / kg, 15 mg / kg and 20 mg / kg, 20 mg / kg and 25 mg / kg, 25 mg / kg and 30 mg / kg, 30 mg / kg and 35 mg / kg, 35 mg / kg and 40 mg / kg, 40 mg / kg and 45 mg / kg, or 45 mg / kg and 50 mg / kg. In some embodiments, the antibody fragment is administered at a dose between 0.3 mg / kg and 10 mg / kg. In some embodiments, the antibody fragment is administered daily, every other day, weekly, biweekly, or monthly. In some embodiments, the antibody fragment is administered at an initial, pre-dosing dose that is higher than the daily, biweekly, weekly, biweekly, or monthly dose. In some embodiments, the initial, pre-dosing dose is between 3 mg / kg and 50 mg / kg. In some embodiments, the initial, pre-dosing dose is between 3 mg / kg and 5 mg / kg, 5 mg / kg and 10 mg / kg, 10 mg / kg and 15 mg / kg, 15 mg / kg and 20 mg / kg, 20 mg / kg and 25 mg / kg, 25 mg / kg and 30 mg / kg, 30 mg / kg and 35 mg / kg, 35 mg / kg and 40 mg / kg, 40 mg / kg and 45 mg / kg, or 45 mg / kg and 50 mg / kg. In some embodiments, the initial, pre-dosing dose is between 3 mg / kg and 20 mg / kg. In some embodiments, the antibody fragment has a shorter half-life compared to its corresponding full-length antibody, e.g., the antibody fragment is rapidly cleared, thereby sparing C1q activity outside the blood space of the subject, or the antibody selectively inhibits C1q within the blood space of the subject, thereby sparing C1q activity outside the blood space of the subject. In some embodiments, the blood space is limited within a blood vessel, such as an artery, arteriole, capillary, venule, or vein. The blood space may contain serum, platelets, endothelial cells, blood cells, or hematopoietic cells.In some embodiments, inhibiting C1q in the blood space of a subject reduces tissue damage in highly vascularized tissues. Examples of highly vascularized tissues are the kidney, alveoli, capillary beds, or glomeruli.

[0337] In some embodiments, the blood disorder is a complement-mediated blood disorder. In some embodiments, the blood disorder is cold agglutinin hemolytic anemia (cold agglutinin disease), cold antibody hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty's disease. The infection may be caused by a thrombocytopenic syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, systemic lupus erythematosus (SLE), glomerulonephritis, antiphospholipid syndrome (APS), an infectious disease, or a drug-induced hematological disorder. The infectious disease may be pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), or a coronavirus. Examples of coronaviruses are selected from SARS-CoV, MERS-CoV, HCoV, HKU1, and SARS-CoV-2. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the subject has a SARS-CoV-2 infection confirmed by reverse transcription-polymerase chain reaction (RT-PCR) from a respiratory tract or blood sample. The blood disorder can be cold agglutinin hemolytic anemia (cold agglutinin disease), warm autoimmune hemolytic anemia (WAIHA), lupus nephritis, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), or immune thrombocytopenic purpura (ITP). Examples of drug-induced hematological disorders are aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, and thrombocytopenia.

[0338] The method promotes improved maintenance of blood cell activation in hematological conditions associated with complement activation. Maintenance of blood function provides functional improvement in hematological disorders relative to untreated patients. A complement inhibitor (e.g., a C1q inhibitor, e.g., an anti-C1q antibody, antibody fragment, and / or antibody derivative) can be administered in an amount and frequency effective to maintain systemic complement inhibition in a subject.

[0339] It is contemplated that the compositions may be obtained and used under the guidance of a physician for in vivo use. The dosage of the therapeutic formulation may vary widely depending on the nature of the disease, the frequency of administration, the mode of administration, clearance of the drug from the host, etc.

[0340] As used herein, "chronically administered," "chronic treatment," "chronically treating," or similar grammatical variants thereof, refers to a treatment regimen used to maintain a certain threshold concentration of a therapeutic agent in the blood of a patient to completely or substantially suppress systemic complement activity in the patient over an extended period of time. Thus, a patient who is chronically treated with a complement inhibitor may be treated chronically for 2 weeks or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; 1, 2 ... or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 12 years or for the remainder of the patient's life). In some embodiments, the complement inhibitor may be administered chronically to a patient in need thereof in an amount and frequency effective to maintain serum hemolytic activity at 20% or less (e.g., less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or even less than 5%). In some embodiments, the complement inhibitor may be administered to a patient in an amount and frequency effective to maintain serum lactate dehydrogenase (LDH) levels within at least 20% of the normal range for LDH (e.g., less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or even 5%).

[0341] In some embodiments, the complement inhibitor is administered to the patient in an amount and frequency effective to maintain a serum LDH level of less than 550 IU / L (e.g., less than 540, 530, 520, 510, 500, 490, 480, 470, 460, 450, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, or less than 270 IU / L). To maintain systemic complement inhibition in the patient, the complement inhibitor can be administered chronically to the patient, for example, once weekly, once every two weeks, twice weekly, daily, monthly, or once every three weeks. In some embodiments of any of the methods described herein, the complement inhibitor (e.g., anti-C1q, anti-C1r, or anti-C1s antibody) can be administered to the patient in an amount and frequency of administration effective to maintain a concentration of bivalent C1q, C1r, or C1s inhibitor molecule(s) (e.g., intact anti-C1q antibody) of at least 0.7 (e.g., at least 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) per C1q molecule in the patient's blood. "Divalent" or "bivalent" with respect to a C1q, C1r, or C1s inhibitor refers to a C1q, C1r, or C1s inhibitor that contains at least two binding sites for a C1q, C1r, or C1s molecule. When the C1q, C1r, or C1s inhibitor is monovalent (e.g., a single-chain anti-C1q, anti-C1r, or anti-C1s antibody or a Fab that binds C1q, C1r, or C1s), the inhibitor can be administered to a patient in an amount and frequency effective to maintain a concentration of at least 1.5 (e.g., at least 2, 2.5, 3, 3.5, 4, 4.5, or 5 or more) monovalent C1q, C1r, or C1s inhibitor per C1q, C1r, or C1s molecule in the blood. In some embodiments, the monovalent C1q, C1r, or C1s inhibitor can be administered to a patient in an amount and frequency effective to maintain a ratio of monovalent C1q, C1r, or C1s inhibitor to C1q, C1r, or C1s of at least 2:1 (e.g., at least 3:1, at least 4:1, at least 5:1, or at least 6:1 or more).In some embodiments, a full (bivalent) anti-C1q, anti-C1r, or anti-C1s antibody is administered to a patient in an amount and frequency effective to maintain a concentration of at least 40 μg (e.g., 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 75, 80, 85, 90, 95, 100, 110, or 120 μg or more) of antibody per milliliter of blood in the patient. In a preferred embodiment, a full anti-C1q, anti-C1r, or anti-C1s antibody is administered in an amount and frequency effective to maintain the antibody at a concentration of at least 50 μg per milliliter of blood in the patient. In preferred embodiments, intact anti-C1q, anti-C1r, or anti-C1s antibodies are administered in an amount and frequency effective to maintain the antibody at a concentration of at least 100 μg per milliliter of blood in the patient. In some embodiments, monovalent anti-C1q, anti-C1r, or anti-C1s antibodies (e.g., single-chain antibodies or Fab fragments) can be administered to a patient in an amount and frequency effective to maintain a concentration of at least 80 μg (e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, or 170 μg or more) of antibody per milliliter of blood in the patient.

[0342] The effective amount of a therapeutic composition given to a particular patient may depend on a variety of factors, some of which may vary from patient to patient. Using ordinary skill, a competent clinician may adjust the dosage of a particular therapeutic or imaging composition during routine clinical testing.

[0343] Therapeutic agents, such as complement inhibitors, gene expression activators, and the like, can be incorporated into a variety of formulations for therapeutic administration by combining with a suitable pharmaceutically acceptable carrier or diluent and formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Thus, administration of the compounds can be achieved in a variety of ways, including oral, buccal, rectal, parenteral, subcutaneous, intraperitoneal, intradermal, transdermal, intrathecal, nasal, intratracheal, and the like. The active agent can be systemic following administration or can be localized by topical administration, the use of intramural administration, or the use of an implant that acts to retain the active dose at the implantation site.

[0344] Combination Treatment The complement inhibitors of the present disclosure may be used in conjunction with any additional treatment, such as, but not limited to, immunosuppressive therapy to treat hematological disorders.

[0345] In some embodiments, the antibodies, antibody fragments, and / or antibody derivatives disclosed herein are administered in combination with inhibitors of the alternative pathway of complement activation. Such inhibitors may include, but are not limited to, factor B inhibitory antibodies, factor D inhibitory antibodies, compstatin-like peptides that block cleavage of soluble, membrane-bound, tagged, or fusion protein forms of CD59, DAF, CR1, CR2, Crry, or C3, non-peptide C3aR antagonists such as SB290157, cobra venom factor, or non-specific complement inhibitors such as nafamostat mesylate (FUTHAN; FUT-175), aprotinin, K-76 monocarboxylic acid (MX-1), and heparin (see, e.g., T. E. Mollnes & M. Kirschfink, Molecular Immunology 43 (2006) 107-121). In some embodiments, the antibodies of the present disclosure are administered in combination with inhibitors of the interaction between an autoantibody and its autoantigen. Such inhibitors may include purified soluble forms of autoantigens or antigen mimics, such as peptide or RNA-derived mimotopes (including mimotopes of AQP4 antigens). Alternatively, such inhibitors may include blocking agents that recognize autoantigens and prevent autoantibody binding without triggering the classical complement pathway. Such blocking agents may include, for example, autoantigen-binding RNA aptamers or antibodies that lack C1q, C1r, or C1s binding sites in the Fc domain (e.g., Fab fragments or antibodies engineered not to bind to C1q, C1r, or C1s).

[0346] In some embodiments, the inhibitors of complement described herein (e.g., inhibitors of C1q, C1r, or C1s, e.g., anti-C1q, anti-C1r, or anti-C1s antibodies or antigen-binding fragments, or antibody derivatives thereof) are used to treat blood disorders (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombocytopenia, It may be formulated with one or more additional active agents useful for treating or ameliorating the symptoms of thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, glomerulonephritis, and / or antiphospholipid syndrome (APS), autoimmune disorders (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), infectious diseases (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), immune complex diseases (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or drug-induced hematological disorders (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin). For example, anti-C1q, anti-C1r, or anti-C1s antibodies can be formulated with antihypertensive agents, anticoagulants, and / or steroids (e.g., corticosteroids). Examples of anticoagulants include, for example, warfarin (Coumadin), aspirin, heparin, phenindione, fondaparinux, idraparinux, and thrombin inhibitors (e.g., argatroban, lepirudin, bivalirudin, or dabigatran).C1q, C1r, or C1s inhibitors (e.g., anti-C1q, anti-C1r, or anti-C1s antibodies) can also be formulated with fibrinolytic agents (e.g., ancrod, ε-aminocaproic acid, antiplasmin-ai, prostacyclin, and defibrotide), cyclophosphamide, or anti-cytokine agents. Anti-cytokine agents include, for example, antibodies or soluble receptors that bind to and modulate the activity of cytokines (e.g., proinflammatory cytokines such as IL-13). In some embodiments, the inhibitors can be formulated with or for use with anti-CD20 agents, such as rituximab (Rituxan™; Biogen, Cambridge, MA). In some embodiments, C1q, C1r, or C1s inhibitors can be formulated for administration to a subject with intravenous immunoglobulin therapy (IVIG) or plasma exchange.

[0347] When an inhibitor of C1q, C1r, or C1s is used in combination (e.g., concomitantly) with a second active agent, or when two or more inhibitors of C1q, C1r, or C1s are used (e.g., anti-C1q, anti-C1r, or anti-C1s antibodies), the agents can be formulated separately or together. For example, the respective pharmaceutical compositions can be mixed, e.g., immediately prior to administration, and administered together, or can be administered separately, e.g., at the same or different times.

[0348] The composition can be formulated with an anti-C1q, anti-C1r, or anti-C1s antibody such that it contains a therapeutically effective amount of an inhibitor of C1q, C1r, or C1s (e.g., an anti-C1q, anti-C1r, or anti-C1s antibody or antigen-binding fragment, or antibody derivative thereof), or the composition can be formulated such that the combined components are effective in treating a blood disorder (e.g., cold agglutinin hemolytic anemia (cold agglutinin disease), hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin Hemolytic anemia, warm antibody hemolytic anemia, warm antibody autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, ABO incompatibility acute hemolytic reaction, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HIT) The compositions may be formulated to comprise a sub-therapeutic amount of the inhibitor and a sub-therapeutic amount of one or more additional active agents so as to be therapeutically effective in treating an autoimmune disorder (e.g., systemic lupus erythematosus (SLE), Crohn's disease, ulcerative colitis), an infectious disease (e.g., pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), coronavirus), an immune complex disease (e.g., cryoglobulinemia, serum sickness, glomerulonephritis), or a drug-induced hematological disorder (e.g., aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, thrombocytopenia from drugs such as penicillin, quinine, or heparin). In some embodiments, compositions can be formulated to contain sub-therapeutic doses of two or more inhibitors of C1q, C1r, or C1s, respectively, such that the inhibitors together provide a therapeutically effective concentration for treating a hematological disorder. Methods for determining a therapeutically effective dose (e.g., a therapeutically effective dose of an anti-C5 antibody) are known in the art and described herein.

[0349] In some embodiments, the antibodies of the present disclosure can be administered in combination with other therapies for blood disorders, for example, the compositions can be administered to a subject simultaneously with, before, or after plasmapheresis, IVIG therapy, plasma infusion, or plasma exchange. [Example]

[0350] Example 1: Anti-C1q antibodies inhibit complement-mediated hemolysis in blood samples from CAD Individual CAD serum samples were pooled together for hemolysis and FACS experiments using increasing concentrations of anti-C1q antibodies. Hemolysis was performed by sensitizing RBCs with pooled CAD serum (10 μL serum + 10 μL RBCs at 4°C for 1 h). Lysis was induced by adding 200 μL of 20x normal human serum for 35 min at 37°C. After lysis, the supernatant was removed, and hRBCs were stained with anti-C3 (CT-C3), anti-C1q, and anti-C4 antibodies for 30 min, washed once, and stained with a fluorescent secondary anti-goat antibody for FACS analysis.

[0351] In CAD, RBCs are coated with C1q, C4b, and C3b, the three major classical complement "opsonins" that induce RBC clearance via "extravascular lysis." C1q, C4b, and C3b are recognized in the spleen and liver by the reticuloendothelial system for RBC removal. Also in CAD, RBCs are coated with C5b, which triggers the formation of the membrane attack complex for direct "intravascular" RBC lysis. Anti-C1q antibodies effectively halt both the intravascular and extravascular RBC lysis processes in CAD serum samples. Anti-C1q inhibits the deposition of all major "opsonins" / immune cell ligands (C1q, C4b, and C3b) of the complement cascade (Figure 1A). Full-length anti-C1q antibodies (e.g., Mab1 antibody comprising the heavy chain variable domain of SEQ ID NO: 33 and the light chain variable domain of SEQ ID NO: 37) and anti-C1s (e.g., TNT009) antibodies inhibit complement-mediated hemolysis (Figure 1B). Anti-C1q antibodies are at least as potent as TNT009 for inhibiting hemolysis (Figure 2A), while anti-C1q antibodies only inhibit the upstream binding of C1q to target cells (Figure 2B). Anti-C1s antibodies do not inhibit C1q binding to RBCs. Selective inhibition of C1q fully inhibits hemolysis induced by the classical pathway while sparing hemolysis induced via the lectin and alternative pathways. In contrast, anti-C5 inhibits the hemolytic activity of all three pathways (Figure 3). Serum biomarkers of complement depletion / consumption in CAD patients provide additional assessment. The decrease in C4 and C2, but not C5, indicates overactivation of the early complement cascade along with consumption of early complement components (Figure 4).CAD can be treated by subcutaneous administration of an anti-C1q antibody (e.g., FabA, an anti-C1q antibody Fab fragment comprising a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40) to inhibit RBC lysis in primates (Figure 5).

[0352] Example 2: Anti-C1q antibodies inhibit hemolysis and complement deposition in blood samples from CAD patients CAD and control plasma samples Human CAD plasma samples from eight subjects were obtained under an IRB-approved protocol. Control serum and plasma samples were obtained from Innovative Research (Novi, MI).

[0353] Ex vivo sensitization of human RBCs Human RBCs (Innovative Research, MI) were washed and suspended in GVB++ buffer (Comptech, TX) (80 μL RBC pack in 2 mL GVB++ buffer). 25 μL of human RBCs were mixed with 25 μL of 5-fold diluted CAD or normal serum and incubated at 4°C for 30 minutes. This step allows cold agglutinin antibodies from CAD subjects to bind to human RBC surface antigens.

[0354] Three subjects showed robust IgG deposits, while seven subjects showed robust IgM deposits, and one subject showed low signals for both cell surface IgG and IgM.

[0355] Hemolysis assay Addition of normal human serum to CAD-sensitized RBCs results in complement mobilization and activation. Normal human serum (20-fold diluted in GVB++ buffer) was added to sensitized human RBCs in GVB++ or GVB-EDTA buffer. For pharmacological studies, anti-C1q antibodies (e.g., Mab2 antibody containing a heavy chain variable domain of SEQ ID NO: 8 and a light chain variable domain of SEQ ID NO: 4) and FabA (e.g., a Fab fragment containing a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40) were titrated into the serum at concentrations ranging from 100 μg / mL to 0.3 μg / mL. RBCs were incubated at 37°C for 30 minutes to allow C1q mobilization and activation of the classical complement cascade in human RBCs.

[0356] Sensitized RBCs incubated in serum diluted with GVB-EDTA buffer (Comptech, TX) served as a negative control because EDTA results in complete inhibition of hemolysis via the complement cascade. RBCs incubated in water served as a positive control to define the maximum lysis possible for each RBC preparation and experimental run.

[0357] After 30 minutes of incubation at 37°C, the cells were spun down in a centrifuge at 2000 rpm for 5 minutes. The supernatant was transferred to a clear-bottom 96-well plate, and hemolysis was quantified by reading the absorbance at 415 nm (hemoglobin-specific absorbance) using a plate reader (Spectramax, CA). To provide an indication of lysis specifically driven by the classical complement cascade, the absorbance signal from wells with serum in GVB-EDTA buffer was subtracted from all other wells. The EDTA-corrected absorbance signal was plotted and evaluated. For pharmacological studies, the signal in each well was also normalized to wells lacking anti-C1q antibody, and the % change in signal was plotted (Figures 6A and 6B). A 4PL-logistic fit was performed to determine the IC values ​​for hemolysis inhibition with anti-C1q MAB2 and FabA. 50 The relative IC for inhibition of hemolysis was determined. 50 was approximately 10 nM for both anti-C1q MAB2 and FabA.

[0358] Flow cytometry to assess complement deposition on human RBCs Human RBCs that were not lysed in the above reaction were washed with dPBS containing 1% BSA and 2 mM EDTA (FACS buffer) and then stained with anti-C4 goat polyclonal antibody (Abcam Ab47788) and anti-C3d-specific polyclonal rabbit antibody (Agilent A0063) for 30 minutes on ice. The cells were then washed with FACS buffer, spun down, and stained with secondary antibodies, anti-goat Alexa 647 conjugate and anti-rabbit Alexa 488 conjugate (Thermo, CA). After 30 minutes of incubation on ice, the cells were washed with FACS buffer and then loaded onto a flow cytometer (Novocyte system, ACEA, CA).

[0359] After the CAD sensitization step, to understand the characteristics of anti-RBC antibodies in individual CAD subjects, RBC cell surface IgG and IgM were detected with respective fluorescently tagged anti-human IgG / IgM antibodies.

[0360] For RBC flow analysis, forward scatter (FSC) and side scatter signals (SSC) were used to identify RBC populations. Single-cell RBC populations were isolated by selecting cells along the diagonal line of an FSC-area vs. FSC-width plot. Single-cell RBCs positive for fluorescent signals in the green (488 nm) and far-red (647 nm) channels were used to define positively labeled cells for cell surface C4 and C3d, respectively. The GVB-EDTA buffer-subtracted % labeled cells for C4 and C3d staining were assessed for differences between CAD and control subjects. For pharmacology studies, the % labeled cells in wells containing MAB2 or FabA were normalized to wells lacking anti-C1q antibody and plotted as percent change (Figures 6A and 6B). A 4PL-logistic fit was performed to determine the IC values ​​for inhibition of C4 and C3d deposition with MAB2 and FabA in these studies. 50 The relative IC for inhibition of complement deposition was determined. 50 was approximately 10 nM for both anti-C1q MAB2 and FabA.

[0361] Example 3: Complement activation by PF4 / heparin via the classical pathway in plasma from patients with heparin-induced thrombocytopenia (HIT) In HIT, RBCs lyse when patients develop antibodies against therapeutically administered heparin in combination with the endogenous circulating protein PF4. To confirm that this lysis is mediated by the classical pathway rather than the alternative pathway, specific chelation studies using EDTA and EGTA were performed in vitro using plasma from HIT patients. The alternative pathway, which is sensitive to Mg2+, is inhibited by EDTA but not EGTA. As shown in Figure 7A, adding EDTA or EGTA to plasma before the addition of PF4 / heparin eliminated complement activation. Furthermore, Mg2+ supplementation of EGTA-treated plasma did not rescue complement activation by PF4 / heparin. Plasma from healthy donors was incubated with or without C1 inhibitor (10 and 20 IU / mL) before incubation with PF4 / heparin, and complement activation by PF4 / heparin was determined by an antigen-C3c capture ELISA assay. As shown in Figure 7B, complement activation was reduced using C1 esterase inhibitors. Similar results were obtained in whole blood assays using flow cytometry (Figures 7C-7D). Whole blood from healthy donors was incubated with or without EDTA (10 mM) or EGTA (10 mM) ± MgCl2 (10 mM), followed by incubation with buffer or antigen (PF4; 25 μg / mL ± heparin; 0.25 U / mL). Binding of PF4 / heparin and C3c to B cells was determined by flow cytometry.

[0362] To investigate the involvement of the lectin and classical pathways, plasma or whole blood from healthy donors was preincubated with various concentrations of a monoclonal antibody against Clq (anti-C1q MAb, Cell Sciences, Inc., Newburyport, MA) or MBL or a mouse isotype control (0–100 μg / mL) before the addition of PF4 / heparin. The complement activation response to PF4 / heparin was assessed by immunoassay (Figure 7E) or flow cytometry (Figures 7F–7G). For flow cytometry experiments, whole blood from healthy donors was incubated with 100 μg / mL of mouse IgG1, anti-MBL, or anti-Clq antibodies, followed by incubation with PF4 / heparin. Binding of PF4 / heparin and C3c to B cells was determined by flow cytometry.

[0363] The anti-Clq MAb inhibited complement activation by PF4 / H in a concentration-dependent manner, whereas the anti-MBL antibody or mouse isotype control did not. Furthermore, data not shown exclude the involvement of the individual lectin proteins, ficolins 2 and 3, in complement activation by the PF4 / heparin complex. The mass spectrometry data accompanying Figure 8 did not correlate the lectin proteins with the complement activation phenotype, nor did functional inhibition of ficolin 2 correlate with loss of complement activation in the immunoassay.

[0364] These studies establish that complement is activated by PF4 / heparin via the classical complement pathway. They also demonstrate that significant donor variation in circulating IgM levels may contribute to host susceptibility to immune activation and provide a target for therapeutic intervention to prevent HIT.

[0365] Example 4: Anti-C1q prevents KKO-induced thrombus formation in a laser microvascular injury model A heparin-induced thrombocytopenia / thrombosis transgenic mouse model expressing both human platelet FcγRIIA and hPF4, described by Reilly et al., Blood. 2001 Oct 15;98(8):2442-7, was used in this study. Anti-C1q antibodies (anti-C1q Mab1, Mab2, and Fab, as well as an isotype control) were intravenously injected into the transgenic mice. The percentage change in thrombus size was measured based on the binding of fluorescently labeled platelets in mice receiving either anti-C1q Mab1, Mab2, Fab, or an isotype control, followed by knockout.

[0366] Example 5: Anti-C1q antibodies inhibit complement deposition in blood samples from AIHA patients Human wAIHA plasma samples from two subjects were obtained under an IRB-approved protocol. Control serum and plasma samples were obtained from Innovative Research, MI.

[0367] Human RBCs (Innovative Research, MI) were suspended in GVB++ buffer (Comptech, TX) (0.5 mL of type O+ single-donor washed RBCs in 10 mL of GVB++ buffer), centrifuged at 2000 rpm for 5 minutes, and the supernatant was decanted. The cells were resuspended to 0.5 mL in GVB++, and 1 mL of 0.5% bromelain (w / v) in dPBS was added. The cells were incubated at 37°C for 10 minutes, then 10 mL of GVB++ buffer was added and centrifuged at 2000 rpm for 5 minutes. The supernatant was decanted, and the cells were resuspended to 0.5 mL in GVB++. A 0.5% RBC solution was made by adding 5 μL of the resuspended cells to 995 μL of GVB++.

[0368] A clear-bottom 96-well plate was used, and in each well, the following reagents were added: healthy donor serum (37.5 μL); 200 μg / mL eculizumab in GVB++ (37.5 μL); patient serum (7.5 μL); GVB++ (42.5 μL) without drug or with MAB2 (1058 μg / mL) for a final concentration of 300 μg / mL; and 0.5% RBCs in GVB++ (25 μL).

[0369] After 2 hours of incubation at 37°C, a flow buffer wash (1% BSA w / v, 2 mM EDTA, dPBS) was added, and the cells were spun down in a centrifuge at 2000 rpm for 5 minutes. The supernatant was removed, and the pellet was resuspended in 100 μL of flow staining solution (1:2000 fluorescein-conjugated anti-C1q (Dako), 1:1500 phycoerythrin-conjugated anti-C3d (Dako), and 1:1000 allophycocyanin-conjugated anti-C4 (Abcam)) and stained for 30 minutes at 4°C in the dark. After incubation, 150 μL of flow buffer wash was added, and the cells were centrifuged at 2000 rpm for 5 minutes. The supernatant was removed, and the cells were resuspended in 125 μL of flow buffer.

[0370] For RBC flow analysis, forward scatter (FSC) and side scatter signals (SSC) were used to identify RBC populations. Single-cell RBC populations were isolated by selecting cells along the diagonal line of an FSC-area vs. FSC-width plot. Single-cell RBCs positive for fluorescent signal in the far-red (647 nm) channel were used to define cells positively labeled for cell surface C4. GVB EDTA samples were used as a negative control for complement deposition. For pharmacology studies, the percentage of labeled cells in wells containing MAB2 was compared to wells lacking anti-C1q antibodies and plotted as a percentage change (Figure 13). This figure shows that sera from patients with wAIHA contain antibodies against RBCs that trigger complement activation and deposition (measured by C4). Mab1 fully prevented C1q activation and C4 deposition.

[0371] Example 6: Clinical trial of anti-C1q monoclonal antibody (MAB1) in patients with warm autoimmune hemolytic anemia (wAIHA). The primary objective of this clinical trial is to evaluate the safety, tolerability, and efficacy of two weekly intravenous infusions of Mab1 (30, 50, 75, or 100 mg / kg) in subjects with warm autoimmune hemolytic anemia (wAIHA).

[0372] Study Design: This is a repeat-dose clinical trial in adult male and female subjects with wAIHA. The study is designed to evaluate the safety, tolerability, and efficacy of Mab1 in subjects with wAIHA. Subjects will receive an IV infusion of Mab1 (30, 50, 75, or 100 mg / kg) on ​​days 1 and 8.

[0373] Methodology: A total of 6-12 subjects with wAIHA will be enrolled in each cohort (i.e., 30, 50, 75, and 100 mg / kg Mab1). All subjects will receive an IV infusion on Day 1 and a second IV infusion on Day 8.

[0374] Screening Visits (Weeks -6 and -2): All subjects undergo study screening procedures within 42 days prior to dosing with Mab1. Screening includes obtaining informed consent, assessing medical history and study eligibility, reviewing vaccination history, baseline health status, administration of the FACIT fatigue questionnaire, and laboratory tests including DAT and markers of hemolysis (reticulocyte count, haptoglobin, LDH, and indirect bilirubin). Study Visits: Subjects receive an intravenous infusion of 30, 50, 75, or 100 mg / kg Mab1 on Days 1 and 8.

[0375] Study assessments for safety, PK, and PD may be completed either in the clinic or at home on Days 3 and 4. Subjects return to the clinic for study assessments for safety, PK, and PD on Days 15, 22, 29, 36, 43, 50, 57, and 71.

[0376] Study Evaluations: Pharmacokinetic parameters will be assessed by serial serum sampling, and pharmacodynamic parameters will be assessed by measurement of CH50 and C4 and other complement biomarkers in the blood, blood cell flow cytometry for complement components, and decline in disease-related biomarkers (e.g., hemoglobin, reticulocyte count, haptoglobin, lactase dehydrogenase, bilirubin, etc.).

[0377] Example 7: Daily subcutaneous dosing of anti-C1q antibody Fab fragment ("FabA") in cynomolgus monkeys Cynomolgus monkeys (2 females / group) were dosed subcutaneously in the interscapular space with anti-C1q antibody Fab fragments (comprising a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40) ("FabA") once a week (5 mg / kg on day 1 and 2 mg / kg for 6 consecutive days). Blood was collected and processed for K2Edta plasma and serum at the following time points: pre-dose, 1, 3, 6, 12, and 24 hours post-dose, and on days 3, 4, 5, 6, 7, 8, 9, and 10. Blood samples on days 2-7 were taken prior to dosing on those days.

[0378] PK and PD ELISA assays: Levels of serum-free FabA (PK), plasma-free C1q (PD), and plasma total C1q (PD) were measured using a sandwich ELISA. Black 96-well plates (Costar #3925) were coated with 75 μL of each capture protein / antibody (Table 1) in bicarbonate buffer (pH 9.4) overnight at 4°C. The next day, the plates were washed with dPBS pH 7.4 (Dulbecco's phosphate-buffered saline) and then blocked with dPBS buffer containing 3% bovine serum albumin (BSA). A standard curve was generated with purified proteins (Table 1) in assay buffer (dPBS containing 0.3% BSA and 0.1% Tween 20). Study serum or plasma samples were prepared at their respective dilutions in assay buffer. The blocking buffer was removed from the plate by tapping. Standards and samples were added in duplicate at 75 μL per well and incubated with shaking at 300 rpm at room temperature for 1 hour for PK measurements, followed by overnight incubation at 4°C, followed by 30 minutes at 37°C and 1 hour at room temperature for the C1q assay. The plate was washed three times with dPBS containing 0.05% Tween 20, and 75 μL of alkaline phosphatase-conjugated secondary antibody (Table 1) was added to all wells. The plate was incubated with shaking at room temperature for 1 hour. The plate was washed three times with dPBS containing 0.05% Tween 20 and developed using 75 μL of alkaline phosphatase substrate (Life Technologies, T2214). After 20 minutes at room temperature, the plate was read using a luminometer. Standards were fitted using a 4PL logistic fit to determine unknown concentrations. Analyte levels were corrected for dilution and then plotted using GraphPad Prism.

[0379] [Table 3]

[0380] Free FabA levels were measured in serum samples from all treated animals (Figure 9). Plasma free C1q levels, which represent the amount of C1q not bound to FabA, were measured in plasma samples from treated animals (Figure 10).

[0381] Ex-vivo hemolysis assay: Serum samples from cynomolgus monkeys were used as a complement source to monitor complement-mediated lysis activity against antibody-sensitized sheep red blood cells (RBCs). Sheep RBCs presensitized with anti-RBC antibody (CompTech #B200) were suspended in gelatinized veronal-buffered saline containing calcium and magnesium (GVB++) (CompTech #B102). RBCs were washed three times with GVB++ and spun down at 2000 rpm for 5 min at 4-6°C to remove any nonspecific signal from the pre-lysed RBD. Cells were resuspended in GVB++ at a final concentration of approximately 200 million cells / mL and kept on ice. Cynomolgus monkey serum samples collected at baseline and after dosing with FabA were diluted 50-fold in GVB++, and 50 μL of each was added to a round-bottom clear plate. The lysis reaction was initiated by adding 50 μL of RBCs to the serum sample and incubated at 37°C for 20 minutes. The plate was then spun down at 2000 rpm for 5 minutes; the supernatant was transferred to a clear, flat-bottom 96-well plate, and the absorbance was read at 415 nm on a plate reader. Control samples were run to estimate background signals using a buffer control without serum or serum samples prepared in GVB buffer containing EDTA. Sample signals were background-subtracted, normalized to baseline, and plotted as a percentage of baseline to determine the time course of hemolysis and the relative inhibition of hemolysis after dosing with FabA.

[0382] Serum hemolysis was inhibited after repeated daily subcutaneous dosing of FabA (FIG. 11).

[0383] Subcutaneous dosing of FabA at 5 mg / kg followed by 2 mg / kg once daily in monkeys resulted in robust PK with measurable free drug levels in both animal groups for at least 1 day after the final dose. Free C1q levels were fully inhibited after the 5 mg / kg dose, and with repeated dosing at 2 mg / kg once daily, free C1q levels were inhibited by 60-90% over the duration of dosing and for at least 1 day after the final dose. Plasma total C1q levels remained unchanged over the duration of the study in both dose groups, suggesting that FabA does not significantly affect C1q turnover. These results confirm that multiple SC dosing of FabA at 2 mg / kg or higher can result in robust free drug levels in the blood and inhibit free C1q and serum hemolytic activity in monkeys.

[0384] Example 8: Evaluation of blood versus tissue distribution of anti-C1q inhibitors This example is used to demonstrate that daily subcutaneous (SC) administration of a defined dose of an anti-C1q inhibitor (e.g., anti-C1q antibody Mab1, Mab2, or FabA) is not sufficient to completely saturate or inhibit C1q in tissue compartments (i.e., the extravascular space) compared to anti-C1q inhibitors delivered by intravenous infusion or injection, but results in complete saturation and inhibition of C1q in the blood (i.e., the intravascular space).

[0385] Animal species. Animal species are first identified in which the anti-C1q inhibitor(s) bind C1q with high affinity and exhibit complete functional inhibition of the classical complement cascade in serum.

[0386] Dose selection of anti-C1q inhibitors: Animals are first treated with anti-C1q FabA (e.g., anti-C1q Fab comprising a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40) at doses of 1, 3, 5, and 10 mg / Kg, and / or anti-C1q monoclonal antibody (e.g., Mab2 antibody comprising a heavy chain variable domain of SEQ ID NO: 8 and a light chain variable domain of SEQ ID NO: 4, or Mab2 antibody comprising a heavy chain variable domain of SEQ ID NO: 33 and a light chain variable domain of SEQ ID NO: 37) at doses of 3, 5, 7, and 10 mg / Kg via a single SC injection. In parallel, additional animals are treated with a comparison molecule (i.e., Mab2 antibody comprising a heavy chain variable domain of SEQ ID NO: 8 and a light chain variable domain of SEQ ID NO: 4, or Mab2 antibody comprising a heavy chain variable domain of SEQ ID NO: 33 and a light chain variable domain of SEQ ID NO: 37) at a dose of 100 mg / Kg IV. Plasma samples are collected at baseline, 30 minutes, 1, 4, 8 hours, and on days 2, 3, 4, 5, and 8. Blood samples are assessed for levels of anti-C1q inhibitor / comparator molecule and inhibition of C1q and serum hemolytic activity. The SC dose at which free drug levels are measurable in blood along with complete inhibition of free C1q for at least 24 hours is determined. IV dosing of a comparative anti-C1q monoclonal antibody at 100 mg / kg results in complete inhibition of C1q for at least 5-8 days after a single dose.

[0387] Tissue distribution of SC doses of anti-C1q inhibitor: Next, animals are treated with a single SC injection of anti-C1q inhibitor at the selected dose, which results in sufficient saturation of C1q in the blood for 24 hours in the initial dose selection study. In parallel, additional animals are treated with a comparison molecule at a dose of 100 mg / Kg IV. Animals are euthanized at 8 hours, 2, 3, and 4 days. Blood is collected at each time point. The animals are then perfused with sterile saline to completely flush blood from the vascular compartment. Tissues, including skin, subcutaneous fat, liver, lung, and muscle, are collected. At each time point, blood samples are evaluated for anti-C1q inhibitor / comparison molecule levels and inhibition of C1q and serum hemolytic activity. Tissue samples (absent blood) are homogenized and evaluated for anti-C1q inhibitor / comparison molecule levels and inhibition of tissue C1q at each time point. A single SC administration of anti-C1q inhibitors shows complete saturation and inhibition of C1q in the blood for 24 hours (up to day 2), but not on days 3 and 4. In tissue samples, free drug levels are below the limit of quantification, and no inhibition of free C1q is observed at any time point. These results indicate that after a single subcutaneous administration of anti-C1q inhibitors, drug levels are measurable in blood but not in tissue samples. Furthermore, C1q is fully inhibited in blood but not in tissue samples.

[0388] Tissue distribution of multiple daily fixed SC doses of anti-C1q inhibitor: Animals are treated with a sing...

Claims

1. 1. A method for preventing, reducing the risk of developing, or treating a blood disorder, comprising administering a C1q inhibitor to a subject.

2. 2. The method of claim 1, wherein the C1q inhibitor is an antibody, an aptamer, an antisense nucleic acid, or a gene editing agent.

3. The method of claim 1 , wherein the inhibitor is an anti-C1q antibody.

4. The method of claim 3, wherein the anti-C1q antibody inhibits the interaction between C1q and autoantibodies, or between C1q and C1r, or between C1q and C1s.

5. The method of claim 3 , wherein the anti-C1q antibody promotes clearance of C1q from the circulation or tissues.

6. The anti-C1q antibody has a dissociation constant (K Ｄ The method according to any one of claims 3 to 5, wherein

7. The method of any one of claims 3 to 6, wherein the anti-C1q antibody binds to C1q with a binding stoichiometry ranging from 20:1 to 1.0:1 or less than 1.0:

1.

8. 8. The method of claim 7, wherein the antibody is an anti-C1q antibody that binds to C1q with a binding stoichiometry ranging from 6:1 to 1.0:1 or less than 1.0:

1.

9. 9. The method of claim 8, wherein the antibody is an anti-C1q antibody that binds to C1q with a binding stoichiometry ranging from 2.5:1 to 1.0:1 or less than 1.0:

1.

10. The method of any one of claims 3 to 9, wherein the antibody specifically binds to C1q and neutralizes its biological activity.

11. 11. The method of claim 10, wherein the biological activity is (1) C1q binding to autoantibodies, (2) C1q binding to C1r, (3) C1q binding to C1s, (4) C1q binding to IgM, (5) C1q binding to phosphatidylserine, (6) C1q binding to pentraxin-3, (7) C1q binding to C-reactive protein (CRP), (8) C1q binding to globular C1q receptor (gC1qR), (9) C1q binding to complement receptor 1 (CR1), (10) C1q binding to beta-amyloid, (11) C1q binding to calreticulin, (12) C1q binding to apoptotic cells, or (13) C1q binding to B cells.

12. The biological activities include (1) activation of the classical complement pathway, (2) reduction in lysis and / or reduction in C3 deposition, (3) activation of antibody and complement dependent cytotoxicity, (4) CH50 hemolysis, (5) reduction in erythrocyte lysis, (6) reduction in erythrocyte phagocytosis, (7) reduction in dendritic cell infiltration, (8) inhibition of complement mediated erythrocyte lysis, (9) reduction in lymphocyte infiltration, (10) reduction in macrophage infiltration, (11) reduction in antibody deposition, (12) reduction in erythrocyte lysis, (13) reduction in erythrocyte phagocytosis, (14) reduction in dendritic cell infiltration, (15) reduction in erythrocyte lysis, (16) reduction in erythrocyte lysis, (17) reduction in erythrocyte lysis, (18) reduction in erythrocyte lysis, (19) reduction in lymphocyte infiltration, (20) reduction in macrophage infiltration, (21) reduction in erythrocyte lysis, (22) reduction in erythrocyte lysis, (23) reduction in erythrocyte lysis, (24) reduction in erythrocyte lysis, (25) reduction in erythrocyte lysis, (26) reduction in erythrocyte phagocytosis, (27) reduction in dendritic cell infiltration, (28) reduction in erythrocyte lysis, (29) reduction in erythrocyte infiltration, (30) reduction in erythrocyte lysis, (31) reduction in erythrocyte lysis, (32) reduction in erythrocyte lysis, (33) reduction in erythrocyte lysis, (34) reduction in erythrocyte lysis, (35) reduction in erythrocyte lysis, (36) reduction in erythrocyte lysis, (37) reduction in erythrocyte lysis (13) reduced neutrophil infiltration, (14) reduced platelet phagocytosis, (15) improved graft survival, (16) reduced macrophage-mediated phagocytosis, (17) reduced autoantibody-mediated complement activation, (18) reduced red blood cell destruction in transfusion reactions, (19) reduced alloantibody-mediated red blood cell lysis, (20) reduced hemolysis in transfusion reactions, (21) reduced alloantibody-mediated platelet lysis, (22) improved anemia, (23) improved graft survival, (24) reduced C3 deposition on red blood cells (e.g., reduced C3b, iC3b, etc. deposition on RBCs); (25) reduced C3 deposition on platelets (e.g., reduced C3b, iC3b, etc. deposition on platelets); (26) reduced anaphylatoxin production; (27) reduced autoantibody-mediated rash formation; (28) reduced autoantibody-induced lupus erythematosus; (29) reduced red blood cell destruction due to transfusion reactions; (30) reduced platelet lysis due to transfusion reactions; (31) reduced mast cell (32) reduced mast cell histamine release, (33) reduced vascular permeability, (34) reduced complement deposition on graft endothelium, (35) B cell antibody production, (36) dendritic cell maturation, (37) T cell proliferation, (38) cytokine production, (39) microglial activation, (40) Arthus reaction, (41) reduced anaphylatoxin production in graft endothelium, or (42) activation of complement receptor 3 (CR3 / C3)-expressing cells.

13. 13. The method of claim 12, wherein the CH50 hemolysate comprises human CH50 hemolysate.

14. The method of claim 12 or 13, wherein the antibody is capable of neutralizing at least about 50% to about 100% of human CH50 hemolysis.

15. 15. The method of any one of claims 12-14, wherein the antibody is capable of neutralizing at least 50% of CH50 hemolysis at a dose of less than 150 ng / ml, less than 100 ng / ml, less than 50 ng / ml, or less than 20 ng / ml.

16. The method of any one of claims 3 to 15, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a human antibody, a chimeric antibody, a monovalent antibody, a multispecific antibody, an antibody fragment, or an antibody derivative thereof.

17. 17. The method of claim 16, wherein the antibody is an antibody fragment, and the antibody fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, or a single-chain antibody molecule.

18. The method of any one of claims 3 to 17, wherein the antibody comprises a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence of SEQ ID NO:

7.

19. The method of any one of claims 3 to 18, wherein the antibody comprises a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO:

11.

20. The method of any one of claims 3 to 19, wherein the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 4 and 35 to 38, and the light chain variable domain comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence of SEQ ID NO:

7.

21. 21. The method of claim 20, wherein the light chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 4 and 35-38.

22. The method of any one of claims 3 to 21, wherein the antibody comprises a heavy chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 8 and 31 to 34, and the heavy chain variable domain comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO:

11.

23. 23. The method of claim 22, wherein the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 8 and 31-34.

24. The method of any one of claims 3 to 23, wherein the antibody is an antibody fragment comprising a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO:

40.

25. The method of any one of claims 3 to 24, wherein the antibody is administered by parenteral injection or infusion.

26. 26. The method of claim 25, wherein the parenteral injection or infusion is subcutaneous or intramuscular.

27. 26. The method of claim 25, wherein the parenteral injection or infusion is an intravenous injection or infusion.

28. The method of any one of claims 3 to 23, wherein the antibody is a full-length antibody.

29. 29. The method of claim 28, wherein the antibody is administered to the subject by intravenous injection or infusion at a dose of between 10 mg / kg and 150 mg / kg.

30. 30. The method of claim 29, wherein the antibody is administered to the subject by intravenous injection or infusion at a dose of between 75 mg / kg and 100 mg / kg.

31. The method of any one of claims 28 to 30, wherein the antibody is administered once a week.

32. The method of any one of claims 28 to 30, wherein the antibody is administered once every two weeks.

33. 31. The method of any one of claims 28 to 30, wherein the antibody is administered monthly.

34. 29. The method of claim 28, wherein the antibody is administered to the subject by subcutaneous or intramuscular injection at a dose of between 1 mg / kg and 10 mg / kg.

35. 35. The method of claim 34, wherein the antibody is administered to the subject by subcutaneous or intramuscular injection at a dose of between 3 mg / kg and 5 mg / kg.

36. 36. The method of claim 34 or 35, wherein the antibody is administered daily.

37. 36. The method of claim 34 or 35, wherein the antibody is administered once every two days.

38. 36. The method of claim 34 or 35, wherein the antibody is administered once a week.

39. 36. The method of claim 34 or 35, wherein the antibody is administered once every two weeks.

40. 36. The method of claim 34 or 35, wherein the antibody is administered monthly.

41. The method of any one of claims 3 to 24, wherein the antibody is an antibody fragment.

42. 42. The method of claim 41, wherein the antibody fragment is administered to the subject by intravenous injection or infusion.

43. 42. The method of claim 41, wherein the antibody fragment is administered to the subject by intramuscular injection.

44. 42. The method of claim 41, wherein the antibody fragment is administered to the subject by subcutaneous injection.

45. 45. The method of any one of claims 41 to 44, wherein the antibody fragment is administered at a dose between 0.1 mg / kg and 50 mg / kg.

46. 46. ​​The method of claim 45, wherein the antibody fragment is administered at a dose between 0.3 mg / kg and 10 mg / kg.

47. The method of any one of claims 41 to 46, wherein the antibody fragment is administered daily.

48. The method of any one of claims 41 to 46, wherein the antibody fragment is administered once every two days.

49. The method of any one of claims 41 to 46, wherein the antibody fragment is administered once a week.

50. The method of any one of claims 41 to 46, wherein the antibody fragment is administered once every two weeks.

51. 47. The method of any one of claims 41 to 46, wherein the antibody fragment is administered once a month.

52. 52. The method of any one of claims 47 to 51, wherein the antibody fragment is administered at an initial preparatory dose that is higher than the daily, every other day, weekly, biweekly, or monthly doses.

53. 53. The method of claim 52, wherein the initial preparatory dose is between 3 mg / kg and 50 mg / kg.

54. 54. The method of claim 53, wherein the initial preparatory dose is between 3 mg / kg and 20 mg / kg.

55. 55. The method of any one of claims 41 to 54, wherein the antibody fragment has a shorter half-life compared to the corresponding full-length antibody.

56. 56. The method of any one of claims 41 to 55, wherein the antibody fragment is rapidly cleared, thereby sparing C1q activity outside the blood space of the subject.

57. 57. The method of any one of claims 41 to 56, wherein the antibody selectively inhibits C1q within the blood space of the subject, thereby sparing C1q activity outside the blood space of the subject.

58. 58. The method of claim 57, wherein the blood cavity is confined within a blood vessel.

59. 59. The method of claim 58, wherein the blood vessel is an artery, arteriole, capillary, venule, or vein.

60. 60. The method of any one of claims 57 to 59, wherein the blood cavity comprises serum, platelets, endothelial cells, blood cells, or hematopoietic cells.

61. 61. The method of any one of claims 57-60, wherein inhibiting C1q in the blood space of the subject reduces tissue damage in highly vascular tissues.

62. 62. The method of claim 61, wherein the highly vascular tissue is a kidney, a pulmonary alveolus, a capillary bed, or a glomerulus.

63. 63. The method of any one of claims 1 to 62, wherein the blood disorder is a complement-mediated blood disorder.

64. The blood disorders include cold agglutinin hemolytic anemia (cold agglutinin disease), cold antibody hemolytic anemia, ABO incompatible acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, neonatal alloimmune thrombocytopenia, red blood cell alloimmunization, Felty syndrome, antibody-mediated thrombocytopenia, Hepatitis B syndrome, and the like.

64. The method of any one of claims 1 to 63, wherein the disorder is heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, systemic lupus erythematosus (SLE), glomerulonephritis, antiphospholipid syndrome (APS), infection, or drug-induced hematological disorder.

65. 65. The method of claim 64, wherein the infectious disease is pneumonia, mycoplasma, mononucleosis, hepatitis C, human immunodeficiency virus (HIV), or coronavirus.

66. 66. The method of any one of claims 65, wherein the coronavirus is selected from SARS-CoV, MERS-CoV, HCoV, HKU1, and SARS-CoV-2.

67. 67. The method of claim 66, wherein the coronavirus is SARS-CoV-2.

68. 68. The method of claim 67, wherein the subject has SARS-CoV-2 infection confirmed by reverse transcription polymerase chain reaction (RT-PCR) from an airway or blood sample.

69. 65. The method of claim 64, wherein the blood disorder is cold agglutinin hemolytic anemia (cold agglutinin disease).

70. 65. The method of claim 64, wherein the blood disorder is warm autoimmune hemolytic anemia (WAIHA).

71. 65. The method of claim 64, wherein the blood disorder is lupus nephritis.

72. 65. The method of claim 64, wherein the blood disorder is heparin-induced thrombocytopenia (HIT).

73. 65. The method of claim 64, wherein the blood disorder is heparin-induced thrombocytopenia and thrombosis (HITT).

74. 65. The method of claim 64, wherein the blood disorder is immune thrombocytopenic purpura (ITP).

75. 65. The method of claim 64, wherein the drug-induced hematological disorder is aplastic anemia, agranulocytosis, megaloblastic anemia, hemolytic anemia, or thrombocytopenia.

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