Humanized anti-complement factor Bb antibodies and uses thereof

Abstract

Provided herein are humanized anti-factor Bb antibodies, methods of producing the antibodies, and methods of using the antibodies.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 012,590, filed April 20, 2020, the entire contents of which are incorporated herein by reference.

[0002] This application relates to humanized anti-factor Bb antibodies and uses of said antibodies. [Background technology]

[0003] The complement system is part of the innate immune system, and its primary role is to "complement" the ability of antibodies and phagocytes to remove harmful pathogens from the organism. The complement system contains three distinct upstream activation pathways (the classical pathway, the alternative pathway, and the lectin pathway), all of which converge into a common terminal pathway. Factor B is a component of the alternative pathway of complement and is cleaved into factors Ba and Bb. Factor B also contains a serine protease (SP) domain, which, when activated, provides the catalytic activity of the alternative pathway C3 and C5 convertases. Abnormal activation of the complement system can cause damage to host tissues in a wide variety of pathological settings, ranging from autoimmune diseases to organ transplantation. There remains a need for treatments for diseases or disorders associated with the complement system. The present invention addresses this need and others. Summary of the Invention [Means for solving the problem]

[0004] In some embodiments, the present disclosure provides humanized anti-factor Bb antibodies, compositions comprising the antibodies, methods for producing the antibodies, and methods for using the antibodies, for example, for treating complement-mediated diseases or disorders. As shown in the data provided herein, the humanized anti-factor Bb antibodies of the present disclosure have binding affinities within two-fold of those of the parent antibody. Unexpectedly, early attempts to humanize the parent mouse anti-factor Bb antibody produced a large number of variants lacking acceptable binding affinity. Thus, multiple rounds of humanization were required to produce humanized versions with appropriate binding affinity (e.g., for treating complement-mediated diseases or disorders). Furthermore, certain V H and V L Domains alone could be combined to produce antibodies that bind to factor Bb with acceptable binding affinity, eg, antibodies tested in Example 1, see Table 9.

[0005] Some embodiments of the present disclosure provide a heavy chain variable region (V) that specifically binds to human complement factor Bb protein and comprises the amino acid sequence of SEQ ID NO: 19. H ) and a light chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 27 L ) is provided.

[0006] Another embodiment of the present disclosure is a V antibody that specifically binds to human complement factor Bb protein and comprises the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L The present invention provides a humanized antibody comprising:

[0007] In some embodiments, the humanized antibody is -6 ~10 -9 It specifically binds to human complement factor Bb protein with an affinity of M.

[0008] In some embodiments, the humanized antibody inhibits complement pathway activity. In some embodiments, the complement pathway activity is alternative pathway (AP) activity.

[0009] In some embodiments, the complement AP activity is selected from the group consisting of AP-mediated terminal membrane attack complex (MAC) deposition, AP-mediated hemolysis, C3 fragment deposition on red blood cells or other cell types, C3b / Bb-mediated cleavage of C3, and C3bBb3b-mediated cleavage of C5.

[0010] In some embodiments, the humanized antibody is a bispecific or multispecific antibody.

[0011] In some embodiments, the humanized antibody is selected from the group consisting of an Ig monomer, a Fab fragment, a F(ab')2 fragment, a scFv, a scAb, and an Fv.

[0012] In some embodiments, the humanized antibody comprises a heavy chain constant region of isotype IgG1, IgG2, IgG3, or IgG4.

[0013] In some embodiments, the humanized antibody comprises an IgG4 constant region or a variant thereof.

[0014] In some embodiments, the heavy chain constant region comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs:28-30.

[0015] In some embodiments, the humanized antibody comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 32-34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0016] In some embodiments, the humanized antibody comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 36-38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0017] Also provided herein are conjugates comprising the humanized antibodies of the present disclosure.

[0018] In some embodiments, the humanized anti-factor Bb antibody of the conjugate comprises a V Hand V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the humanized anti-factor Bb antibody of the conjugate comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:34 and a light chain comprising the amino acid sequence of SEQ ID NO:35.

[0019] In some embodiments, the humanized anti-factor Bb antibody of the conjugate comprises a V H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the humanized anti-factor Bb antibody of the conjugate comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:38 and a light chain comprising the amino acid sequence of SEQ ID NO:39.

[0020] Further provided herein is a pharmaceutical composition comprising a humanized antibody described herein or a conjugate described herein.

[0021] In some embodiments, the pharmaceutical composition comprises a VIII antibody comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the pharmaceutical composition comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0022] In some embodiments, the pharmaceutical composition comprises a VIII antibody comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the pharmaceutical composition comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0023] In some embodiments, the pharmaceutical composition comprises a VIII antibody comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 LIn some embodiments, the pharmaceutical composition comprises a conjugate comprising a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0024] In some embodiments, the pharmaceutical composition comprises a VIII antibody comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the pharmaceutical composition comprises a conjugate comprising a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0025] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0026] Also provided herein is a device comprising a humanized antibody described herein, a conjugate described herein, or a pharmaceutical composition described herein.

[0027] In some embodiments, the device comprises a V H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the device comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0028] In some embodiments, the device comprises a V H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the device comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0029] In some embodiments, the device comprises a V H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the device comprises a conjugate comprising a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0030] In some embodiments, the device comprises a V H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the device comprises a conjugate comprising a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0031] In some embodiments, the device comprises a V H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the device comprises a pharmaceutical composition comprising a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0032] In some embodiments, the device comprises a V H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the device comprises a pharmaceutical composition comprising a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0033] In some embodiments, the device is an injection device, such as a syringe, a pen, or an electronic injection device (e-device).

[0034] Yet another aspect of the present disclosure provides a method of treating a subject having a complement-mediated disease or disorder, the method comprising administering to the subject an effective amount of a humanized antibody described herein, a conjugate described herein, or a pharmaceutical composition described herein to treat the complement-mediated disease or disorder.

[0035] In some embodiments, the method of treating a subject having a complement-mediated disease or disorder includes administering to a subject a V antibody comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the method of treating a subject having a complement-mediated disease or disorder comprises administering to the subject an effective amount of a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35, to treat the complement-mediated disease or disorder.

[0036] In some embodiments, the method of treating a subject having a complement-mediated disease or disorder includes administering to a subject a V antibody comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, a method of treating a subject having a complement-mediated disease or disorder comprises administering to the subject an effective amount of a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39 to treat the complement-mediated disease or disorder.

[0037] In some embodiments, the method of treating a subject having a complement-mediated disease or disorder includes administering to a subject a V antibody comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 LIn some embodiments, the method of treating a subject having a complement-mediated disease or disorder comprises administering to the subject an effective amount of a conjugate comprising a humanized anti-factor Bb antibody comprising:

[0038] In some embodiments, the method of treating a subject having a complement-mediated disease or disorder includes administering to a subject a V antibody comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the method of treating a subject having a complement-mediated disease or disorder comprises administering to the subject an effective amount of a conjugate comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39 to treat the complement-mediated disease or disorder.

[0039] In some embodiments, the method of treating a subject having a complement-mediated disease or disorder includes administering to a subject a V antibody comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the method of treating a subject having a complement-mediated disease or disorder comprises administering to the subject an effective amount of a pharmaceutical composition comprising a humanized anti-factor Bb antibody comprising:

[0040] In some embodiments, the method of treating a subject having a complement-mediated disease or disorder includes administering to a subject a V antibody comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 LIn some embodiments, the method of treating a subject having a complement-mediated disease or disorder comprises administering to the subject an effective amount of a pharmaceutical composition comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39 to treat the complement-mediated disease or disorder.

[0041] In some embodiments, the complement-mediated disease is selected from the group consisting of IgA nephropathy (Berge's disease), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), lupus nephritis, ANCA vasculitis, membranous nephropathy, C3 glomerulonephritis (C3GN), focal segmental glomerulosclerosis (FSGS), multiple sclerosis, macular degeneration, age-related macular degeneration (AMD), rheumatoid arthritis, antiphospholipid syndrome, asthma, ischemia-reperfusion injury, type II membranoproliferative glomerulonephritis (GN), spontaneous abortion, pauci-immune vasculitis, epidermolysis bullosa, recurrent abortion, and traumatic brain injury.

[0042] Yet another aspect of the present disclosure provides a method for inhibiting complement pathway activity in a subject. In some embodiments, the complement pathway activity is alternative pathway (AP) activity. In some embodiments, the method for inhibiting complement pathway activity (e.g., AP activity) in a subject comprises administering to the subject an effective amount of a humanized anti-factor Bb antibody, a conjugate comprising a humanized anti-factor Bb antibody, or a pharmaceutical composition comprising a humanized anti-factor Bb antibody to inhibit complement pathway activity.

[0043] In some embodiments, the method of inhibiting complement pathway activity (e.g., AP activity) in a subject includes administering to a subject a V polypeptide comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 LIn some embodiments, a method for inhibiting complement pathway activity (e.g., AP activity) in a subject comprises administering to the subject an effective amount of a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35, thereby inhibiting complement pathway activity.

[0044] In some embodiments, the method of inhibiting complement pathway activity (e.g., AP activity) in a subject includes administering to a subject a V polypeptide comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, a method for inhibiting complement pathway activity (e.g., AP activity) in a subject comprises administering to the subject an effective amount of a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39, thereby inhibiting complement pathway activity.

[0045] In some embodiments, the method of inhibiting complement pathway activity (e.g., AP activity) in a subject includes administering to a subject a V polypeptide comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, a method for inhibiting complement pathway activity (e.g., AP activity) in a subject comprises administering to the subject an effective amount of a conjugate comprising a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35, thereby inhibiting complement pathway activity.

[0046] In some embodiments, the method of inhibiting complement pathway activity (e.g., AP activity) in a subject includes administering to a subject a V polypeptide comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 LIn some embodiments, a method for inhibiting complement pathway activity (e.g., AP activity) in a subject comprises administering to the subject an effective amount of a conjugate comprising a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39, thereby inhibiting complement pathway activity.

[0047] In some embodiments, the method of inhibiting complement pathway activity (e.g., AP activity) in a subject includes administering to a subject a V polypeptide comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, a method for inhibiting complement pathway activity (e.g., AP activity) in a subject comprises administering to the subject an effective amount of a pharmaceutical composition comprising a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35, thereby inhibiting complement pathway activity.

[0048] In some embodiments, the method of inhibiting complement pathway activity (e.g., AP activity) in a subject includes administering to a subject a V polypeptide comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, a method for inhibiting complement pathway activity (e.g., AP activity) in a subject comprises administering to the subject an effective amount of a pharmaceutical composition comprising a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39, thereby inhibiting complement pathway activity.

[0049] In some embodiments, the complement AP activity is selected from the group consisting of AP-mediated terminal membrane attack complex (MAC) deposition, AP-mediated hemolysis, C3 fragment deposition on red blood cells or other cell types, C3b / Bb-mediated cleavage of C3, and C3bBb3b-mediated cleavage of C5. In some embodiments, the subject has a complement-mediated disease or disorder.

[0050] In some embodiments, the method further comprises administering a therapeutic agent to the subject.

[0051] In some embodiments, administration is intravenous, subcutaneous, or intramuscular.

[0052] Also provided herein is a humanized anti-factor Bb antibody for use in a method for treating a complement-mediated disease or disorder. Further provided herein is a conjugate comprising a humanized anti-factor Bb antibody for use in a method for treating a complement-mediated disease or disorder. Still further provided herein is a pharmaceutical composition comprising a humanized anti-factor Bb antibody for use in a method for treating a complement-mediated disease or disorder. Still further provided herein is a device comprising a humanized anti-factor Bb antibody for use in a method for treating a complement-mediated disease or disorder.

[0053] In some embodiments, the humanized anti-factor Bb antibody for use in a method for treating a complement-mediated disease or disorder comprises a VB comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, a humanized anti-factor Bb antibody for use in a method for treating a complement-mediated disease or disorder comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:34 and a light chain comprising the amino acid sequence of SEQ ID NO:35.

[0054] In some embodiments, the humanized anti-factor Bb antibody for use in a method for treating a complement-mediated disease or disorder comprises a VB comprising the amino acid sequence of SEQ ID NO: 17. Hand V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, a humanized anti-factor Bb antibody for use in a method for treating a complement-mediated disease or disorder comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:38 and a light chain comprising the amino acid sequence of SEQ ID NO:39.

[0055] In some embodiments, the conjugate for use in the method for treating a complement-mediated disease or disorder comprises a V comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, a conjugate for use in a method for treating a complement-mediated disease or disorder comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0056] In some embodiments, the conjugate for use in the method for treating a complement-mediated disease or disorder comprises a V comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, a conjugate for use in a method for treating a complement-mediated disease or disorder comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:38 and a light chain comprising the amino acid sequence of SEQ ID NO:39.

[0057] In some embodiments, the pharmaceutical composition for use in the method for treating a complement-mediated disease or disorder comprises a V comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, a pharmaceutical composition for use in a method for treating a complement-mediated disease or disorder comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0058] In some embodiments, the pharmaceutical composition for use in the method for treating a complement-mediated disease or disorder comprises a V comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, a pharmaceutical composition for use in a method for treating a complement-mediated disease or disorder comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0059] In some embodiments, the device for use in the method for treating a complement-mediated disease or disorder comprises a V comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, a device for use in a method for treating a complement-mediated disease or disorder comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0060] In some embodiments, the device for use in the method for treating a complement-mediated disease or disorder comprises a V comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, a device for use in a method for treating a complement-mediated disease or disorder comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0061] In some embodiments, the complement-mediated disease is selected from the group consisting of IgA nephropathy (Berge's disease), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), lupus nephritis, ANCA vasculitis, membranous nephropathy, C3 glomerulonephritis (C3GN), focal segmental glomerulosclerosis (FSGS), multiple sclerosis, macular degeneration, age-related macular degeneration (AMD), rheumatoid arthritis, antiphospholipid syndrome, asthma, ischemia-reperfusion injury, membranoproliferative GN type II, spontaneous abortion, microimmune vasculitis, epidermolysis bullosa, recurrent abortion, and traumatic brain injury.

[0062] Also provided herein is a humanized anti-factor Bb antibody for use in a method for inhibiting complement pathway activity (e.g., AP activity). Further provided herein is a conjugate comprising a humanized anti-factor Bb antibody for use in a method for inhibiting complement pathway activity (e.g., AP activity). Still further provided herein is a pharmaceutical composition comprising a humanized anti-factor Bb antibody for use in a method for inhibiting complement pathway activity (e.g., AP activity). Still further provided herein is a device comprising a humanized anti-factor Bb antibody for use in a method for inhibiting complement pathway activity (e.g., AP activity).

[0063] In some embodiments, a humanized anti-factor Bb antibody for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a VBG comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, a humanized anti-factor Bb antibody for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0064] In some embodiments, a humanized anti-factor Bb antibody for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a VBG comprising the amino acid sequence of SEQ ID NO: 17. Hand V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, a humanized anti-factor Bb antibody for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:38 and a light chain comprising the amino acid sequence of SEQ ID NO:39.

[0065] In some embodiments, the conjugate for use in the method for inhibiting complement pathway activity (e.g., AP activity) comprises a V comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, a conjugate for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0066] In some embodiments, the conjugate for use in the method for inhibiting complement pathway activity (e.g., AP activity) comprises a V comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, a conjugate for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0067] In some embodiments, the pharmaceutical composition for use in the method for inhibiting complement pathway activity (e.g., AP activity) comprises a V protein comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 LIn some embodiments, a pharmaceutical composition for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0068] In some embodiments, the pharmaceutical composition for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a V protein comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, a pharmaceutical composition for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0069] In some embodiments, the pharmaceutical composition for use in the method for inhibiting complement pathway activity (e.g., AP activity) comprises a V protein comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, a pharmaceutical composition for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0070] In some embodiments, the pharmaceutical composition for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a V protein comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, a pharmaceutical composition for use in a method for inhibiting complement pathway activity (e.g., AP activity) comprises a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0071] Also provided herein are nucleic acids or nucleic acid sets that encode or collectively encode the humanized antibodies described herein; vectors or vector sets that include the nucleic acids or nucleic acid sets described herein; and cells that express the humanized antibodies, nucleic acids or nucleic acid sets, or vectors or vector sets described herein.

[0072] In some embodiments, the nucleic acid or set of nucleic acids comprises the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the nucleic acid or set of nucleic acids encodes or collectively encodes a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0073] In some embodiments, the nucleic acid or set of nucleic acids comprises the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the nucleic acid or set of nucleic acids encodes or collectively encodes a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0074] In some embodiments, the vector or vector set comprises a V vector comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the vector or vector set comprises a nucleic acid or set of nucleic acids that encode, or collectively encode, a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0075] In some embodiments, the vector or vector set comprises a V vector comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the vector or vector set comprises a nucleic acid or set of nucleic acids that encode, or collectively encode, a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0076] In some embodiments, the cells comprise a V H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the cells comprise a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0077] In some embodiments, the cells comprise a V H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the cells comprise a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0078] In some embodiments, the cells comprise a V H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the cell comprises a nucleic acid or set of nucleic acids that encode, or collectively encode, a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0079] In some embodiments, the cells comprise a V H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the cell comprises a nucleic acid or set of nucleic acids that encode, or collectively encode, a humanized anti-factor Bb antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0080] In some embodiments, the cells comprise a V H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the cell comprises a vector or vector set comprising a nucleic acid or set of nucleic acids that encode, or collectively encode, a humanized anti-factor Bb antibody comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0081] In some embodiments, the cells comprise a V H and V comprising the amino acid sequence of SEQ ID NO: 26 L In some embodiments, the cell comprises a vector or vector set comprising a nucleic acid or set of nucleic acids that encode, or collectively encode, a humanized anti-factor Bb antibody comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

[0082] In some embodiments, the cell is a mammalian cell, for example, selected from the group consisting of a human embryonic kidney (HEK) cell, a Chinese hamster ovary (CHO) cell, an NS0 myeloma cell, an SP2 cell, a COS cell, and a mammary epithelial cell.

[0083] Another aspect of the present disclosure provides a method of producing a humanized antibody described herein, comprising culturing a cell described herein to produce the humanized antibody. In some embodiments, the method further comprises isolating the humanized antibody.

[0084] In some embodiments, the method for producing a humanized anti-factor Bb antibody comprises a V antibody comprising the amino acid sequence of SEQ ID NO: 19. H and V comprising the amino acid sequence of SEQ ID NO: 27 L In some embodiments, the method for producing a humanized anti-factor Bb antibody comprises culturing cells comprising a nucleic acid encoding, or a set of nucleic acids collectively encoding, a humanized anti-factor Bb antibody comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO: 34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35, to produce the humanized anti-factor Bb antibody.

[0085] In some embodiments, the method for producing a humanized anti-factor Bb antibody comprises a V antibody comprising the amino acid sequence of SEQ ID NO: 17. H and V comprising the amino acid sequence of SEQ ID NO: 16 L and culturing cells comprising a nucleic acid encoding, or a set of nucleic acids collectively encoding, a humanized anti-factor Bb antibody comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO:28 and a light chain comprising the amino acid sequence of SEQ ID NO:39, to produce the humanized anti-factor Bb antibody. In some embodiments, the method of producing a humanized anti-factor Bb antibody comprises culturing cells comprising a nucleic acid encoding, or a set of nucleic acids collectively encoding, a humanized anti-factor Bb antibody comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO:28 and a light chain comprising the amino acid sequence of SEQ ID NO:39.

[0086] The above summary is intended to illustrate, without limitation, some of the embodiments, advantages, configurations, and uses of the technology disclosed herein. Other embodiments, advantages, configurations, and uses of the technology disclosed herein will be apparent from the detailed description, drawings, examples, and claims.

[0087] The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component illustrated in various figures is represented by a like numeral. For clarity, not every component is labeled in every figure. [Brief explanation of the drawings]

[0088] [Figure 1A] Figure 1 shows the binding of humanized antibody variants (Group 1) to complement factor Bb (Factor Bb) using single-cycle kinetics. Raw sensorgrams and fitted curves (1:1 binding model) are shown for humanized variant and control antibody binding to Factor Bb. Kinetic analysis was performed on a Biacore T200. Each antibody was captured on a Protein A CM5 chip, after which increasing concentrations of Factor Bb were injected and single off-rates were determined. *The scale is the same for each section, x is -200 to 1400, and y is -5 to 40. [Figure 1B] Figure 1 shows the binding of humanized antibody variants (Group 1) to complement factor Bb (Factor Bb) using single-cycle kinetics. Raw sensorgrams and fitted curves (1:1 binding model) are shown for humanized variant and control antibody binding to Factor Bb. Kinetic analysis was performed on a Biacore T200. Each antibody was captured on a Protein A CM5 chip, after which increasing concentrations of Factor Bb were injected and single off-rates were determined. *The scale is the same for each section, x is -200 to 1400, and y is -5 to 40. [Figure 1C] Figure 1 shows the binding of humanized antibody variants (Group 1) to complement factor Bb (Factor Bb) using single-cycle kinetics. Raw sensorgrams and fitted curves (1:1 binding model) are shown for humanized variant and control antibody binding to Factor Bb. Kinetic analysis was performed on a Biacore T200. Each antibody was captured on a Protein A CM5 chip, after which increasing concentrations of Factor Bb were injected and single off-rates were determined. *The scale is the same for each section, x is -200 to 1400, and y is -5 to 40. [Figure 1D] Figure 1 shows the binding of humanized antibody variants (Group 1) to complement factor Bb (Factor Bb) using single-cycle kinetics. Raw sensorgrams and fitted curves (1:1 binding model) are shown for humanized variant and control antibody binding to Factor Bb. Kinetic analysis was performed on a Biacore T200. Each antibody was captured on a Protein A CM5 chip, after which increasing concentrations of Factor Bb were injected and single off-rates were determined. *The scale is the same for each section, x is -200 to 1400, and y is -5 to 40. [Figure 2A] Figure 1 shows a graph depicting the binding of humanized variants (group 2) to factor Bb using single-cycle kinetics. Raw sensorgrams and fitted curves (1:1 binding model) for the redesigned variants and control antibodies binding to factor Bb are shown. Kinetic analysis was performed on a Biacore T200. Each antibody was captured on a Protein A CM5 chip, after which increasing concentrations of factor Bb were injected and single off-rates were determined. [Figure 2B] Figure 1 shows a graph depicting the binding of humanized variants (group 2) to factor Bb using single-cycle kinetics. Raw sensorgrams and fitted curves (1:1 binding model) for the redesigned variants and control antibodies binding to factor Bb are shown. Kinetic analysis was performed on a Biacore T200. Each antibody was captured on a Protein A CM5 chip, after which increasing concentrations of factor Bb were injected and single off-rates were determined. [Figure 2C] Figure 1 shows a graph depicting the binding of humanized variants (group 2) to factor Bb using single-cycle kinetics. Raw sensorgrams and fitted curves (1:1 binding model) for the redesigned variants and control antibodies binding to factor Bb are shown. Kinetic analysis was performed on a Biacore T200. Each antibody was captured on a Protein A CM5 chip, after which increasing concentrations of factor Bb were injected and single off-rates were determined. [Figure 3]SDS-PAGE gel of Protein A / Post-SEC purified antibodies. 1 μg of each reduced antibody sample was loaded onto a NuPage 4-12% Bis-Tris gel (ThermoFisher, Loughborough, UK) and run at 200 V for 35 minutes. The gel was stained with InstantBlue (Expedeon, Swavesey, UK). Mk:PAGERuler™ Plus pre-stained protein ladder (ThermoFisher, Loughborough, UK). [Figure 4A] 4A-4C are graphs showing binding of lead antibodies (all from group 2) to factor Bb using multi-cycle kinetics. Multi-cycle sensorgram data and fitted curves (1:1 binding model) are shown for humanized variant binding to factor Bb (FIG. 4A: chimeric VH0 / VK0, VH4 / Vκ6; FIG. 4B: VH4 / Vκ7, VH6 / Vκ6; FIG. 4C: VH6 / Vκ7, VH7 / Vκ7). [Figure 4B] 4A-4C are graphs showing binding of lead antibodies (all from group 2) to factor Bb using multi-cycle kinetics. Multi-cycle sensorgram data and fitted curves (1:1 binding model) are shown for humanized variant binding to factor Bb (FIG. 4A: chimeric VH0 / VK0, VH4 / Vκ6; FIG. 4B: VH4 / Vκ7, VH6 / Vκ6; FIG. 4C: VH6 / Vκ7, VH7 / Vκ7). [Figure 4C] 4A-4C are graphs showing binding of lead antibodies (all from group 2) to factor Bb using multi-cycle kinetics. Multi-cycle sensorgram data and fitted curves (1:1 binding model) are shown for humanized variant binding to factor Bb (FIG. 4A: chimeric VH0 / VK0, VH4 / Vκ6; FIG. 4B: VH4 / Vκ7, VH6 / Vκ6; FIG. 4C: VH6 / Vκ7, VH7 / Vκ7). [Figure 5]Figure 1 shows a graph depicting a Factor Bb competitive enzyme-linked immunosorbent assay (ELISA) of the humanized variants against the parent antibody. A dilution series of the anti-Factor Bb variants was tested against a fixed concentration of the murine parent antibody for binding to Factor Bb. Bound murine antibody was detected using an anti-mouse peroxidase conjugate and tetramethylbenzidine (TMB) substrate. [Figure 6A] 6A-6C are graphs showing the activity of WIESLAB® humanized anti-factor Bb antibodies in alternative complement pathway (CAP) (FIG. 6A) and AP-mediated hemolysis (FIGS. 6B-6C) using human serum. [Figure 6B] 6A-6C are graphs showing the activity of WIESLAB® humanized anti-factor Bb antibodies in alternative complement pathway (CAP) (FIG. 6A) and AP-mediated hemolysis (FIGS. 6B-6C) using human serum. [Figure 6C] 6A-6C are graphs showing the activity of WIESLAB® humanized anti-factor Bb antibodies in alternative complement pathway (CAP) (FIG. 6A) and AP-mediated hemolysis (FIGS. 6B-6C) using human serum. [Figure 7A] 7A and 7B are graphs showing the specificity of lead humanized variants for the active form (factor Bb) of human factor B (FIGS. 7A and 7B) or cynomolgus factor B (FIGS. 7C and 7D) by surface plasmon resonance. [Figure 7B] 7A and 7B are graphs showing the specificity of lead humanized variants for the active form (factor Bb) of human factor B (FIGS. 7A and 7B) or cynomolgus factor B (FIGS. 7C and 7D) by surface plasmon resonance. [Figure 7C] 7A and 7B are graphs showing the specificity of lead humanized variants for the active form (factor Bb) of human factor B (FIGS. 7A and 7B) or cynomolgus factor B (FIGS. 7C and 7D) by surface plasmon resonance. [Figure 7D] 7A and 7B are graphs showing the specificity of lead humanized variants for the active form (factor Bb) of human factor B (FIGS. 7A and 7B) or cynomolgus factor B (FIGS. 7C and 7D) by surface plasmon resonance. [Figure 8]Figure 8A shows the specificity of binding of chimeric parent antibody VH0 / VK0-IgG4v1 (Figure 8A) and representative humanized variant antibody VH6 / VK7-IgG4v2 (Figure 8B) to only factor Bb among various complement proteins. [Figure 9] Figure 9B is a graph showing the activity of VH6 / VK7-IgG4v2 (produced from HEK cells) and VH6 / VK7-IgG4v2_CHO (produced from CHO cells) in the WIESLAB® alternative complement pathway (CAP) assay using normal human (Figure 9A) and cynomolgus monkey (Cyno) (Figure 9B) serum. [Figure 10] Figure 10 is a graph showing the activity of VH6 / VK7-IgG4v2 (produced from HEK) and VH6 / VK7-IgG4v2_CHO in the WIESLAB® classical complement pathway (CCP) assay using normal human (Figure 10A) and cynomolgus monkey (Figure 10B) serum. [Figure 11] 1 is a graph showing hemolysis of rabbit RBCs by VH6 / VK7-IgG4v2_HEK and VH6 / VK7-IgG4v2_CHO in normal human serum. [Figure 12] Graph showing affinity and multi-cycle sensorgram raw data with fitted curves for VH6 / VK7-IgG4v2_CHO (left) and VH6 / VK7-IgG4v2_HEK (right) binding to human Bb protein. DETAILED DESCRIPTION OF THE INVENTION

[0089] The present disclosure provides humanized antibodies that bind to complement factor Bb protein. These antibodies are referred to herein as "humanized anti-factor Bb antibodies." The present disclosure also provides nucleic acids encoding the humanized anti-factor Bb antibodies, compositions comprising the antibodies, methods of producing the antibodies (e.g., recombinant production methods), and methods of using the antibodies, such as methods of treating (at least one) complement-mediated disease or disorder.

[0090] "Antibody" encompasses antibodies or immunoglobulins of any isotype, including, but not limited to, humanized antibodies and chimeric antibodies. Antibodies can be single-chain antibodies (scAbs) or single-domain antibodies (dAbs) (e.g., single-domain heavy chain antibodies or single-domain light chain antibodies; see Holt et al. (2003) Trends Biotechnol. 21:484). The term "antibody" also encompasses fragments of antibodies (antibody fragments) that retain specific binding to an antigen. An "antibody" is a fragment of an antibody consisting of two antibody heavy chains (V, V ... H ) and light chain (V L ), as well as single-chain variable fragments (scFv), which are fusion proteins of the variable regions of V connected by a small peptide linker. H and V L (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)). Other fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein are also encompassed by the term "antibody."

[0091] An "antibody fragment" comprises a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include antigen-binding fragment (Fab), Fab', F(ab')2, variable domain Fv fragment (Fv), Fd fragment, and antigen-binding fragment of a chimeric antigen receptor.

[0092] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a name reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0093] "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and antigen-binding site. This region comprises a dimer of one heavy-chain and one light-chain variable domain in tight, non-covalent association. The three CDRs of each variable domain interact to form a V H -V L It is in this configuration that defines an antigen-binding site on the surface of the dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv, comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0094] A "Fab" fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including at least one cysteine ​​from the antibody hinge region. Fab'-SH is the designation herein for Fab', in which the cysteine ​​residues of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0095] "scFv" antibody fragments are V fragments of an antibody H and V L and these regions are present in a single polypeptide chain. In some cases, an Fv polypeptide comprises a V H and V L The scFv further comprises a polypeptide linker between the domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York, pp. 269-315 (1994).

[0096] "Diabody" refers to a small antibody fragment with two antigen-binding sites, the fragments being attached to the same polypeptide chain, V L V connected to H (V H -V L ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain and create two antigen-binding sites. Diabodies are more fully described, for example, in Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).

[0097] Antibodies can be monovalent or bivalent. Antibodies can be Ig monomers, which are "Y-shaped" molecules consisting of four polypeptide chains: two heavy chains and two light chains connected by disulfide bonds.

[0098] The antibody may be detectably labeled, for example, with a radioisotope, an enzyme that generates a detectable product, and / or a fluorescent protein. The antibody may be further conjugated to other moieties, such as a member of a specific binding pair, for example, the biotin member of the biotin-avidin specific binding pair. The antibody may also be bound to a solid support, including, but not limited to, a polystyrene plate and / or beads.

[0099] An "isolated" antibody is one that has been identified and separated and / or removed from a component of its natural environment (i.e., not occurring in nature). Contaminant components of its natural environment are materials that would interfere with the antibody's use (e.g., diagnostic or therapeutic use), and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some cases, the antibody is purified (1) to greater than 90%, greater than 95%, or greater than 98%, e.g., greater than 99%, by weight of the antibody as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions using Coomassie blue or silver staining. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. In some embodiments, isolated antibody is prepared by at least one purification step.

[0100] A "monoclonal antibody" is an antibody produced by a population of identical cells, all of which are produced from a single cell by repeated cell replication. That is, a clone of cells produces only a single antibody species. Monoclonal antibodies are produced using hybridoma production techniques, although other production methods known to those skilled in the art can also be used (e.g., antibodies derived from antibody phage display libraries).

[0101] "Complementarity-determining regions (CDRs)" are non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. CDRs are described by Lefranc et al. (2003) Developmental and Comparative Immunology 27:55; Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and 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 variant thereof is intended to be within the scope of the term as defined and used herein.

[0102] 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.

[0103] "Framework," when used in reference to an antibody variable region, includes all amino acid residues outside the CDR regions in the variable region of an antibody. A variable region framework is generally a discontinuous amino acid sequence that includes only those amino acids outside the CDRs. A "framework region" includes each domain of the framework that is separated by a CDR.

[0104] A "humanized antibody" is an antibody containing portions of antibodies of different origins, at least one portion of which contains an amino acid sequence of human origin. For example, a humanized antibody can contain a portion derived from an antibody of non-human origin, such as a mouse, with the requisite specificity, and a portion derived from an antibody sequence of human origin, chemically joined together by conventional techniques (e.g., synthesis) or prepared as a contiguous polypeptide using genetic engineering techniques (e.g., DNA encoding the protein portion of the chimeric antibody is expressed to produce a contiguous polypeptide chain) (e.g., chimeric immunoglobulin). Another example of a humanized antibody is an antibody containing at least one chain containing CDRs derived from an antibody of non-human origin and framework regions derived from a light chain and / or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes). Chimeric or CDR-grafted single-chain antibodies are also encompassed by the term humanized immunoglobulin. See, for example, Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694 B1; Neuberger, MS et al., International Publication No. WO 86 / 01533; Neuberger, MS et al., European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Padlan, EA et al., European Patent Application No. 0,519,596 A1. Regarding single chain antibodies, see also Ladner et al., US Pat. No. 4,946,778; Huston, US Pat. No. 5,476,786; and Bird, RE et al., Science 242:423-426 (1988).

[0105] In some embodiments, humanized antibodies are produced using synthetic and / or recombinant nucleic acids to prepare genes (e.g., cDNAs) encoding the desired humanized chains. For example, nucleic acid (e.g., DNA) sequences encoding humanized variable regions can be constructed using PCR mutagenesis methods to alter the DNA sequence encoding the human or humanized chain, such as a DNA template from a previously humanized variable region (see, e.g., Kamman, M. et al., Nucl. Acids Res., 17:5404 (1989); Sato, K. et al., Cancer Research, 53:851-856 (1993); Daugherty, BL et al., Nucleic Acids Res., 19(9):2471-2476 (1991); and Lewis, AP and JSCrowe, Gene, 101:297-302 (1991)). Variants can also be readily produced using these or other suitable methods. For example, cloned variable regions can be mutagenized and sequences encoding variants with desired specificities can be selected (e.g., from phage libraries; see, e.g., Krebber et al., U.S. Pat. No. 5,514,548; Hoogenboom et al., WO 93 / 06213, published April 1, 1993).

[0106] Humanized anti-factor Bb antibody The amino acid sequences of murine monoclonal anti-factor Bb antibodies from the humanized anti-factor Bb antibodies described herein are provided in Table 1. In some embodiments, the humanized anti-factor Bb antibodies comprise heavy and / or light chain variable region framework regions that comprise sequences derived from a human immunoglobulin framework.

[0107] [Table 1]

[0108] In some embodiments, the humanized anti-factor Bb antibodies described herein comprise heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), and heavy chain complementarity determining region 3 (CDR-H3) of the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the humanized anti-factor Bb antibodies described herein comprise light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3) of the light chain variable region comprising the amino acid sequence of SEQ ID NO: 13. In some embodiments, the humanized anti-factor Bb antibodies further comprise a humanized heavy chain framework region and / or a humanized light chain framework region.

[0109] In some embodiments, according to the Kabat definition, the humanized anti-factor Bb antibodies described herein comprise a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, according to the Kabat definition, the humanized anti-factor Bb antibodies described herein comprise a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the humanized anti-factor Bb antibodies further comprise a humanized heavy chain framework region and / or a humanized light chain framework region. In some embodiments, according to the IMGT definition, the humanized anti-factor Bb antibodies described herein comprise a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 8, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, according to the IMGT definition, the humanized anti-factor Bb antibodies described herein comprise a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. In some embodiments, the humanized anti-factor Bb antibodies further comprise a humanized heavy chain framework region and / or a humanized light chain framework region.

[0110] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises the V H V contains at most 20 amino acid variations (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to H In some embodiments, the anti-factor Bb antibody of the present disclosure comprises the V L V contains at most 20 amino acid variations (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to L Includes.

[0111] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises the V H V comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to H In some embodiments, the humanized anti-Bb factor of the present disclosure comprises the V L V comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to L Includes.

[0112] In some embodiments, a humanized anti-factor Bb antibody of the present disclosure comprises a CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 1, a CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 2, a CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 3, and a V as set forth in SEQ ID NO: 12. H humanized V containing at most 20 amino acid variations in the framework regions (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to HIn some embodiments, a humanized anti-factor Bb antibody of the present disclosure comprises a CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 4, a CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 6, and a V set forth in SEQ ID NO: 13. L humanized V containing at most 20 amino acid variations in the framework regions (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to L Includes.

[0113] In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure comprise a humanized VH1 having the amino acid sequence of SEQ ID NO: 1 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 3 (according to the Kabat definition system). H Including V H The framework regions of V are collectively set forth in SEQ ID NO: 12. H In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising a CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 4, a CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 6. L Including V L The framework regions of V are collectively represented by any one of SEQ ID NOs: 13. L and is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the framework regions of

[0114] In some embodiments, a humanized anti-factor Bb antibody of the present disclosure comprises a CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 7, a CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 8, a CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 9, and a V as set forth in SEQ ID NO: 12. H humanized V containing at most 20 amino acid variations in the framework regions (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to H In some embodiments, a humanized anti-factor Bb antibody of the present disclosure comprises a CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 10, a CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 11, and a CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 6, and a V set forth in SEQ ID NO: 13. L humanized V containing at most 20 amino acid variations in the framework regions (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to L Includes.

[0115] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VH1 having the amino acid sequence of SEQ ID NO: 7 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to the IMGT definition system). H Including V H The framework regions of V are collectively set forth in SEQ ID NO: 12. HIn some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody having a CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 10, a CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 11, and a CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 6. L Including V L The framework regions of V are collectively represented by any one of SEQ ID NOs: 13. L and is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the framework regions of

[0116] Examples of amino acid sequences and DNA coding sequences for the humanized heavy chain variable regions of the humanized anti-factor Bb antibodies described herein are provided in Table 2. Examples of amino acid sequences and DNA coding sequences for the humanized light chain variable regions of the humanized anti-factor Bb antibodies described herein are provided in Table 3.

[0117] [Table 2-1] [Table 2-2]

[0118] [Table 3-1] [Table 3-2]

[0119] In some embodiments, a humanized anti-factor Bb antibody of the present disclosure comprises a CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 1, a CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 2, a CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 3, and a V-seq set forth in any one of SEQ ID NOs: 14 to 20. H humanized V containing at most 20 amino acid variations in the framework regions (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to H In some embodiments, a humanized anti-factor Bb antibody of the present disclosure comprises a CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 4, a CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 6, and a V set forth in any one of SEQ ID NOs: 21 to 27. L humanized V containing at most 20 amino acid variations in the framework regions (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to L Includes.

[0120] In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure comprise a humanized VH1 having the amino acid sequence of SEQ ID NO: 1 (according to the Kabat definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 2 (according to the Kabat definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 3 (according to the Kabat definition system). H Including V H The framework regions of V are collectively represented by any one of SEQ ID NOs: 14 to 20. HIn some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising a CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 4, a CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 6. L Including V L The framework regions of V are collectively set forth in any one of SEQ ID NOs: 21 to 27. L and is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the framework regions of

[0121] In some embodiments, a humanized anti-factor Bb antibody of the present disclosure comprises a CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 7, a CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 8, a CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 9, and a V-seq set forth in any one of SEQ ID NOs: 14 to 20. H humanized V containing at most 20 amino acid variations in the framework regions (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to H In some embodiments, a humanized anti-factor Bb antibody of the present disclosure comprises a CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 10, a CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 11, and a CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 6, and a V set forth in any one of SEQ ID NOs: 21 to 27. Lhumanized V containing at most 20 amino acid variations in the framework regions (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to L Includes.

[0122] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VH1 having the amino acid sequence of SEQ ID NO: 7 (according to the IMGT definition system), a CDR-H2 having the amino acid sequence of SEQ ID NO: 8 (according to the IMGT definition system), and a CDR-H3 having the amino acid sequence of SEQ ID NO: 9 (according to the IMGT definition system). H Including V H The framework region of V is set forth in any one of SEQ ID NOs: 14 to 20. H In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody having a CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 10, a CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 11, and a CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 6. L Including V L The framework regions of V are collectively set forth in any one of SEQ ID NOs: 21 to 27. L and is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the framework regions of

[0123] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody set forth in any one of SEQ ID NOs: 14-20. H In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VBV sequence set forth in any one of SEQ ID NOs: 21-27. L Table 4 includes the humanized V H and one of the humanized V provided in Table 3 LExamples of humanized anti-factor Bb antibodies are provided, including one of:

[0124] [Table 4-1] [Table 4-2]

[0125] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising the amino acid sequence of SEQ ID NO: 19. H and a humanized V comprising the amino acid sequence of SEQ ID NO: 27 L Includes.

[0126] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising the amino acid sequence of SEQ ID NO: 17. H and a humanized V comprising the amino acid sequence of SEQ ID NO: 26 L Includes.

[0127] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising the amino acid sequence of SEQ ID NO: 19. H and a humanized V comprising the amino acid sequence of SEQ ID NO: 26 L Includes.

[0128] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising the amino acid sequence of SEQ ID NO: 17. H and a humanized V comprising the amino acid sequence of SEQ ID NO: 27 L Includes.

[0129] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising the amino acid sequence of SEQ ID NO: 20. H and a humanized V comprising the amino acid sequence of SEQ ID NO: 27 L Includes.

[0130] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising the amino acid sequence of SEQ ID NO: 14. Hand a humanized V comprising any one of the amino acid sequences of SEQ ID NO: 21. L Includes.

[0131] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising the amino acid sequence of SEQ ID NO: 14. H and a humanized V comprising any one of the amino acid sequences of SEQ ID NO: 22. L Includes.

[0132] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising the amino acid sequence of SEQ ID NO: 14. H and a humanized V comprising any one of the amino acid sequences of SEQ ID NO: 23. L Includes.

[0133] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising the amino acid sequence of SEQ ID NO: 14. H and a humanized V comprising any one of the amino acid sequences of SEQ ID NO: 24. L Includes.

[0134] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure comprises a humanized VB antibody comprising the amino acid sequence of SEQ ID NO: 14. H and a humanized V comprising any one of the amino acid sequences of SEQ ID NO: 25. L Includes.

[0135] In some embodiments, the humanized anti-factor Bb antibodies described herein are full-length IgG, Ig monomers, Fab fragments, F(ab')2 fragments, scFv, scAb, or Fv. In some embodiments, the humanized anti-factor Bb antibodies described herein are full-length IgG. In some embodiments, the heavy chain of any of the humanized anti-factor Bb antibodies described herein comprises a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can be from any suitable origin, such as human, mouse, rat, or rabbit. In some embodiments, the heavy chain constant region is from a human IgG (gamma heavy chain), such as IgG1, IgG2, or IgG4.

[0136] In some embodiments, mutations may be introduced into the heavy chain constant region of any one of the humanized anti-factor Bb antibodies described herein. In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the heavy chain constant region (numbered according to the Kabat numbering system (e.g., the EU index in Kabat), e.g., in the CH2 domain (residues 231-340 of human IgG1) and / or CH3 domain (residues 341-447 of human IgG1) and / or hinge region) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activating Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that decrease or increase the affinity of the antibody for the Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in antibody Fc receptors that can be made to alter the affinity of the antibody for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and WO 02 / 060919; 98 / 23289; and 97 / 34631, which are incorporated herein by reference.

[0137] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the heavy chain constant region (CH1 domain) to alter (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region, as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered to, for example, facilitate association of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0138] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant domain or its FcRn-binding fragment to alter (e.g., decrease or increase) the half-life of the antibody in vivo. In some embodiments, one or more mutations are introduced into the Fc or hinge-Fc domain fragment. For example, for examples of mutations that may alter (e.g., decrease or increase) the half-life of an antibody in vivo, see, for example, WO 02 / 060919; 98 / 23289; and 97 / 34631; and U.S. Patent Nos. 5,869,046; 6,121,022; 6,277,375; and 6,165,745.

[0139] In some embodiments, the constant region described herein is an IgG1 constant region and comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as per Kabat. See U.S. Patent No. 7,658,921, incorporated herein by reference. This type of mutant IgG, termed "YTE mutant," has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al. (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three, or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat. Additional mutations introduced into the heavy chain constant region that will increase the half-life of the antibody are known in the art, e.g., the M428L / N434S (EU numbering; M459L / N466S Kabat numbering) mutation described in Zalevsky et al., Nat Biotechnol. 2010 Feb;28(2):157-159.

[0140] In some embodiments, one, two, or more amino acid substitutions are introduced into the IgG constant domain Fc region to alter the effector function of the antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (by point mutation or other means) can reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. See, for example, U.S. Patent Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant domains, thereby increasing tumor localization. In some embodiments, at least one amino acid substitution may be introduced in the Fc region of an antibody described herein to remove a potential glycosylation site in the Fc region that may reduce Fc receptor binding (see, e.g., Shields RL et al. (2001) J Biol Chem 276:6591-604).

[0141] In some embodiments, at least one amino acid in the constant region may be replaced with a different amino acid residue so that the antibody has altered Clq binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogie et al.). In some embodiments, at least one amino acid residue in the N-terminal region of the CH2 domain of the antibody described herein is altered to thereby alter the antibody's ability to bind complement. This approach is further described in WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or increase the antibody's affinity for Fcγ receptors. This approach is further described in WO 00 / 42072.

[0142] In some embodiments, to avoid potential complications due to Fab-arm exchange, which is known to occur with natural IgG4 mAbs, the antibodies provided herein may contain a stabilizing "Adair" mutation, in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence (Angal S. et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993). In some embodiments, to reduce residual antibody-dependent cellular cytotoxicity, an L235E (EU numbering, equivalent to L248E in Kabat numbering) mutation is introduced in the heavy chain constant region, as described, for example, in Benhnia et al., JOURNAL OF VIROLOGY, December 2009, pp. 12355-12367.

[0143] In some embodiments, the heavy chain constant region in any one of the humanized anti-factor Bb antibodies described herein is an IgG4 constant region or a variant thereof. Examples of IgG4 constant regions and variants are provided in Table 5.

[0144] [Table 5]

[0145] In some embodiments, the light chain of any of the humanized anti-factor Bb antibodies described herein comprises a light chain constant region (C L In some examples, C L is a kappa light chain. L is a lambda light chain. L is the kappa light chain, the sequence of which is provided below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 31)

[0146] Other antibody heavy and light chain constant regions are known in the art, such as those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0147] In some embodiments, the humanized anti-factor Bb antibodies described herein are selected from the V listed in Table 2. H or any variant thereof, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to any one of SEQ ID NOs: 28-30. H or any variant thereof, and a heavy chain constant region containing at most 20 amino acid variations (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to any one of SEQ ID NOs: 28-30. H or any variant thereof, and a heavy chain constant region comprising the amino acid sequence of any one of SEQ ID NOs: 28 to 30.

[0148] In some embodiments, the humanized anti-factor Bb antibodies described herein are selected from the V listed in Table 3. Lor any variant thereof, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 31. In some embodiments, the humanized anti-factor Bb antibodies described herein comprise a light chain comprising any one of the V listed in Table 3. L or any variant thereof, and a light chain constant region containing at most 20 amino acid variations (e.g., at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to SEQ ID NO: 31. In some embodiments, the humanized anti-factor Bb antibodies described herein comprise a light chain comprising any one of the V listed in Table 3. L or any variant thereof, and a light chain constant region comprising the amino acid sequence of SEQ ID NO:31.

[0149] Exemplary heavy and light chain amino acid sequences of the humanized anti-factor Bb antibodies described herein are provided in Table 6.

[0150] [Table 6-1] [Table 6-2]

[0151] In some embodiments, the humanized anti-factor Bb antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:32 and a light chain comprising the amino acid sequence of SEQ ID NO:35.

[0152] In some embodiments, the humanized anti-factor Bb antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:33 and a light chain comprising the amino acid sequence of SEQ ID NO:35.

[0153] In some embodiments, the humanized anti-factor Bb antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:34 and a light chain comprising the amino acid sequence of SEQ ID NO:35.

[0154] In some embodiments, the humanized anti-factor Bb antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:36 and a light chain comprising the amino acid sequence of SEQ ID NO:39.

[0155] In some embodiments, the humanized anti-factor Bb antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:37 and a light chain comprising the amino acid sequence of SEQ ID NO:39.

[0156] In some embodiments, the humanized anti-factor Bb antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:38 and a light chain comprising the amino acid sequence of SEQ ID NO:39.

[0157] In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure bind to factor Bb protein (e.g., factor Bb protein from a mammal, fish, or invertebrate that has a complement system). In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure bind to a mammalian factor Bb protein. In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure bind to a human factor Bb protein. In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure bind to a factor Bb protein having the amino acid sequence of SEQ ID NO:40.

[0158] Homo sapiens factor Bb protein (SEQ ID NO: 40) KIVLDPSGSMNIYLVLDGSDSIGASNFTGAKKCLVNLIEKVASYGVKPRYGLVTYATYPKIWVKVSEADSSNADWVTKQLNEINYEDHKLKSGTNTKKALQAVYSMMSWPDDVPPEGWNRTRHVII LMTDGLHNMGGDPITVIDEIRDLLYIGKDRKNPREDYLDVYVFGVGPLVNQVNINALASKKDNEQHVFKVKDMENLEDVFYQMIDESQSLSLCGMVWEHRKGTDYHKQPWQAKISVIRPSKGHESC MGAVVSEYFVLTAAHCFTVDDKEHSIKVSVGGEKRDLEIEVVLFHPNYNINGKKEAGIPEFYDYDVALIKLKNKLKYGQTIRPICLPCTEGTTRALRLPPTTTCQQQKEELLPAQDIKALFVSEEE KKLTRKEVYIKNGDKKGSCERDAQYAPGYDKVKDISEVVTPRFLCTGGVSPYADPNTCRGDSGGPLIVHKRSRFIQVGVISWGVVDVCKNQKRQKQVPAHARDFHINLFQVLPWLKEKLQDEDLGFL

[0159] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure has a saturation of about 10 -6 ~10 -11 nM, e.g., about 10 -6 M~about 10 -7 M, about 10 -7 M~about 10 -8 M, about 10 -8 M~about 10 -9 M, about 10 -9 M~about 10 -10 M, or about 10 -10 M~about 10 -11 It binds to complement Bb protein with an affinity of M. The term "about" preceding a numerical value means ±10% of the recited value.

[0160] In some embodiments, humanized anti-factor Bb antibodies of the present disclosure exhibit preferential binding to factor Bb compared to binding to factor B. In some embodiments, humanized anti-factor Bb antibodies of the present disclosure bind to factor Bb but do not substantially bind to soluble factor B. In some embodiments, humanized anti-factor Bb antibodies of the present disclosure bind to factor Bb with an affinity that is at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7.5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least 75-fold, or at least 100-fold higher than the antibody's affinity for factor B. In some embodiments, a humanized anti-factor Bb antibody of the present disclosure binds to factor Bb with an affinity that is 2-fold to 2.5-fold, 2.5-fold to 5-fold, 5-fold to 10-fold, 10-fold to 15-fold, 15-fold to 20-fold, 20-fold to 25-fold, 25-fold to 50-fold, 50-fold to 75-fold, or 75-fold to 100-fold higher than the affinity of the antibody for factor B.

[0161] In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit complement pathway activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the level of complement activity in the absence of the humanized anti-factor Bb antibody.

[0162] In some embodiments, a humanized anti-factor Bb antibody of the present disclosure inhibits alternative pathway (AP) activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the level of AP activity in the absence of the humanized anti-factor Bb antibody. -7 M~10 -9 M IC 50, e.g. 10 -7 M~5×10 -7 M, 5 x 10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5 x 10 -8 M~10 -9 M IC 50 In some embodiments, the complement AP activity is selected from the group consisting of AP-mediated terminal membrane attack complex (MAC) deposition, AP-mediated hemolysis, C3 fragment deposition on red blood cells or other cell types, C3b / Bb-mediated cleavage of C3, and C3bBb3b-mediated cleavage of C5. In some embodiments, the inhibition of complement AP activity by a humanized anti-factor Bb antibody is measured using a Complement System Alternative Pathway WIESLAB® kit.

[0163] In some embodiments, a humanized anti-factor Bb antibody of the present disclosure inhibits the formation of membrane attack complexes (MAC) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the amount of MAC formed in the absence of the anti-factor Bb antibody.

[0164] In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit C3b / Bb-mediated cleavage of C3. C3b / Bb is also known as "C3 convertase." In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit C3b / Bb-mediated cleavage of C3 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to cleavage of C3 in the absence of the anti-factor Bb antibody. ... -7 M~10-9 M IC 50 , e.g. 10 -7 M~5×10 -7 M, 5 x 10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5 x 10 -8 M~10 -9 M IC 50 inhibits C3b / Bb-mediated cleavage of C3.

[0165] In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit C3b / Bb-mediated cleavage of C3, thereby reducing the production of C3 cleavage products. For example, the humanized anti-factor Bb antibodies of the present disclosure may inhibit C3b / Bb-mediated cleavage of C3, thereby reducing the production of C3 cleavage products (e.g., C3a and / or C3b) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the production of C3 cleavage products in the absence of the anti-factor Bb antibody.

[0166] In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit complement AP-mediated cytolysis by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the extent of cytolysis in the absence of the anti-factor Bb antibody. Cytolysis assays can be used to determine the extent of inhibition of AP-mediated cytolysis. In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit complement AP-mediated cytolysis by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the extent of cytolysis in the absence of the anti-factor Bb antibody. -7 M~10 -9 M IC 50 , e.g. 10 -7 M~5×10 -7 M, 5 x 10 -7 M~10 -8 M, 10 -8M~5×10 -8 M, or 5 x 10 -8 M~10 -9 M IC 50 inhibits AP-mediated cell lysis.

[0167] In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit complement AP-mediated hemolysis by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the extent of hemolysis in the absence of the anti-factor Bb antibody. A rabbit red blood cell (RBC) hemolysis assay may be used to determine the extent of inhibition of AP-mediated hemolysis. In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit complement AP-mediated hemolysis by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the extent of hemolysis in the absence of the anti-factor Bb antibody. -7 M~10 -9 M IC 50 , e.g. 10 -7 M~5×10 -7 M, 5 x 10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5 x 10 -8 M~10 -9 M IC 50 inhibits AP-mediated hemolysis.

[0168] In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit AP-mediated deposition of C3b, C3d, or other C3 degradation products on cells or tissues. For example, the humanized anti-factor Bb antibodies of the present disclosure can inhibit AP-mediated deposition of C3b, C3d, or other C3 degradation products on cells or tissues by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, compared to the amount of C3b, C3d, or other C3 degradation products deposited on cells or tissues in the absence of administration of the anti-factor Bb antibody or prior to administration of the anti-factor Bb antibody. In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit AP-mediated deposition of C3b, C3d, or other C3 degradation products on cells or tissues by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. -7 M~10 -9 M IC 50 , e.g. 10 -7 M~5×10 -7 M, 5 x 10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5 x 10 -8 M~10 -9 M IC 50 and inhibits AP-mediated deposition of C3b, C3d, or other C3 degradation products on cells or tissues.

[0169] In some embodiments, a humanized anti-factor Bb antibody of the present disclosure inhibits AP-mediated C3b deposition on a cell or tissue by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the amount of C3b deposition on the cell or tissue in the absence of the humanized anti-factor Bb antibody. -7 M~10 -9 M IC 50 , e.g. 10 -7 M~5×10 -7M, 5 x 10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5 x 10 -8 M~10 -9 M IC 50 and inhibits AP-mediated C3b deposition on cells or tissues.

[0170] In some embodiments, a humanized anti-factor Bb antibody of the present disclosure inhibits AP-mediated C3b deposition on red blood cells (RBCs) or other cell types by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the amount of C3b deposition on RBCs in the absence of the humanized anti-factor Bb antibody. -7 M~10 -9 M IC 50 , e.g. 10 -7 M~5×10 -7 M, 5 x 10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5 x 10 -8 M~10 -9 M IC 50 and inhibits AP-mediated C3b deposition on RBCs.

[0171] In some embodiments, a humanized anti-factor Bb antibody of the present disclosure, when administered to a subject in need thereof, reduces the amount of circulating factor Bb in the subject. For example, a humanized anti-factor Bb antibody of the present disclosure, when administered to a subject in need thereof, may reduce the amount of circulating factor Bb in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to the amount of circulating factor Bb in the subject in the absence of administration of the humanized anti-factor Bb antibody, or compared to the amount of circulating factor Bb in the subject before administration of the humanized anti-factor Bb antibody.

[0172] In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit C3bBb3b-mediated cleavage of C5. In some embodiments, the humanized anti-factor Bb antibodies of the present disclosure inhibit C3bBb3b-mediated cleavage of C5 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to C3bBb3b-mediated cleavage of C5 in the absence of the humanized anti-factor Bb antibody.

[0173] In some embodiments, the humanized anti-factor Bb antibody of the present disclosure is a bispecific or multispecific antibody. For example, the humanized anti-factor Bb antibody may be a bispecific antibody comprising a first antigen-binding portion that specifically binds to an epitope in the complement Bb protein and a second antigen-binding portion that binds to a second antigen.

[0174] Immunoconjugates In some embodiments, a humanized anti-factor Bb antibody of the present disclosure is conjugated to another agent to form an immunoconjugate. For example, a humanized anti-factor Bb antibody may contain a free thiol (—SH) group at the carboxyl terminus, which may be used to attach the antibody to a second polypeptide (e.g., another antibody, including a humanized anti-factor Bb antibody), a scaffold, a carrier, or the like. In some embodiments, a humanized anti-factor Bb antibody comprises at least one non-naturally occurring amino acid. In some embodiments, the non-naturally occurring amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group. See, e.g., U.S. Patent No. 7,632,924 for a description of suitable non-naturally occurring amino acids. Inclusion of a non-naturally occurring amino acid may provide for linkage to a polymer, a second polypeptide, a scaffold, or the like. For example, a humanized anti-factor Bb antibody linked to a water-soluble polymer can be prepared by reacting a water-soluble polymer (e.g., PEG) containing a carbonyl group with an antibody, where the antibody contains a non-naturally encoded amino acid containing an aminooxy, hydrazine, hydrazide, or semicarbazide group. In some embodiments, a humanized anti-factor Bb antibody linked to a water-soluble polymer can be prepared by reacting an antibody containing an alkyne-containing amino acid with a water-soluble polymer (e.g., PEG) containing an azide moiety. In some embodiments, the azide or alkyne group is linked to the PEG molecule through an amide linkage.

[0175] In some embodiments, the humanized anti-factor Bb antibody is linked (e.g., covalently linked) to a polymer (e.g., a polymer other than a polypeptide). Suitable polymers include, for example, biocompatible polymers and water-soluble biocompatible polymers. Suitable polymers include synthetic polymers and naturally occurring polymers. Suitable polymers may have an average molecular weight in the range of 500 Da to 50,000 Da, e.g., 5,000 Da to 40,000 Da or 25,000 to 40,000 Da.

[0176] In some embodiments, the humanized anti-factor Bb antibody comprises a "radiopaque" label, e.g., a label that is easily visualized, for example, using an x-ray. Radiopaque materials are well known to those skilled in the art. The most common radiopaque materials include iodide, bromide, or barium salts. Other radiopaque materials are also known, including, but not limited to, organobismuth derivatives (see, e.g., U.S. Pat. No. 5,939,045), radiopaque multiurethanes (see, e.g., U.S. Pat. No. 5,346,981), organobismuth hybrids (see, e.g., U.S. Pat. No. 5,256,334), and / or radiopaque barium multimer complexes (see, e.g., U.S. Pat. No. 4,866,132).

[0177] In some embodiments, the humanized anti-factor Bb antibody is covalently linked to a second moiety (e.g., a lipid, a polypeptide other than the humanized anti-factor Bb antibody, a synthetic polymer, and / or a carbohydrate) using, for example, glutaraldehyde, a homobifunctional crosslinker, or a heterobifunctional crosslinker.

[0178] In some embodiments, the humanized anti-factor Bb antibody is immobilized on a solid support. Suitable supports are well known in the art and include, among others, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, duracite, wells of reaction trays (e.g., multiwell plates), plastic tubes, and the like. Solid supports can comprise any of a variety of materials, including, for example, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose, and magnetite. Suitable methods for immobilizing humanized anti-factor Bb antibodies on solid supports are well known and include, but are not limited to, ionic, hydrophobic, and / or covalent interactions. Solid supports can be soluble or insoluble, for example, in aqueous solutions. In some embodiments, suitable solid supports are generally insoluble in aqueous solutions.

[0179] In some embodiments, the humanized anti-factor Bb antibody comprises a detectable label. Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Suitable labels include magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, red fluorescent protein, and / or yellow fluorescent protein), radioactive labels (e.g., 3 H, 125 I, 35 S, 14 C, or 32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and others commonly used in enzyme-linked immunosorbent assays (ELISAs)), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0180] In some embodiments, the humanized anti-factor Bb antibody comprises an imaging agent or radioisotope that is suitable for use in imaging, e.g., imaging procedures performed on humans. Non-limiting examples of labels include: 123 I (iodine), 18 F (fluorine), 99 Tc (technetium), 111 In (indium), and 67 Radioactive isotopes such as Ga (gallium), and contrast agents such as gadolinium (Gd), dysprosium, and iron. 153Radioisotopes (Gd) can also be used and are suitable for imaging in non-human mammals. Humanized anti-factor Bb antibodies can be labeled using standard techniques. For example, humanized anti-factor Bb antibodies can be iodinated using chloramine T or 1,3,4,6-tetrachloro-3α,6α-diphenylglycouril. For fluorination, fluorine is added to the humanized anti-factor Bb antibody during synthesis by a fluoride ion displacement reaction. For reviews of protein synthesis using such radioisotopes, see Muller-Gartner, H., TIB Tech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst., 16(2):209-244 (1999). Humanized anti-factor Bb antibodies can also be labeled with imaging agents using standard techniques. For example, a humanized anti-factor Bb antibody can be labeled with Gd by conjugating the antibody with a small Gd chelate, such as Gd-diethylenetriaminepentaacetic acid (GdDTPA) or Gd-tetraazacyclododecanetetraacetic acid (GdDOTA). See Caravan et al., Chem. Rev. 99:2293-2352 (1999) and Lauffer et al., J. Magn. Reson. Imaging, 3:11-16 (1985). For example, a humanized anti-factor Bb antibody can be labeled with Gd by conjugating the antibody with polylysine-Gd chelate. See, for example, Curtet et al., Invest. Radiol., 33(10):752-761 (1998). Alternatively, in some embodiments, a humanized anti-factor Bb antibody can be labeled with Gd by incubating a paramagnetic polymeric liposome containing a Gd chelator lipid with avidin and a biotinylated antibody. See, e.g., Sipkins et al., Nature Med., 4:623-626 (1998).

[0181] Suitable fluorescent proteins include green fluorescent protein (GFP) or variants thereof, blue fluorescent variants of GFP (BFP), cyan fluorescent variants of GFP (CFP), yellow fluorescent variants of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, and Monster. Phycobiliproteins and phycobiliprotein conjugates, including, but not limited to, GFP, paGFP, maple protein and kindling protein, B-phycoerythrin, R-phycoerythrin, and allophycocyanin, are also suitable for use. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, and / or mPlum (Shaner et al. (2005) Nat. Methods 2:905-909). Suitable for use are any of the various fluorescent and colored proteins from anthozoan species, as described, for example, in Matz et al. (1999) Nature Biotechnol. 17:969-973.

[0182] In some embodiments, the humanized anti-factor Bb antibody is conjugated to a therapeutic agent. Any of the humanized anti-factor Bb antibodies disclosed herein can be used to form an antibody-agent conjugate. The agent can be attached to the N-terminus of the light chain, the C-terminus of the light chain, the N-terminus of the heavy chain, or the C-terminus of the heavy chain. In some embodiments, the agent is attached to the hinge of the antibody or to at least one other site on the antibody. For single-chain antibodies, the agent can be attached to the N- or C-terminus of the single-chain antibody. The agent can be conjugated to the antibody directly or via a linker using techniques known to those skilled in the art. The linker can be cleavable or non-cleavable. Examples of such therapeutic agents (e.g., for use in therapy) are known to those skilled in the art.

[0183] In some embodiments, the humanized anti-factor Bb antibody is linked (eg, covalently or non-covalently linked) to a fusion partner, such as a ligand; an epitope tag; a peptide; and / or a protein other than an antibody. Suitable fusion partners include peptides and polypeptides that confer enhanced in vivo stability (e.g., increased serum half-life); provide ease of purification, e.g., (His)n, e.g., 6His; provide secretion of the fusion protein from cells; provide epitope tags, e.g., GST, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO: 55), FLAG (e.g., DYKDDDDK; SEQ ID NO: 56), and / or c-myc (e.g., EQKLISEEDL; SEQ ID NO: 57); provide a detectable signal, e.g., an enzyme that generates a detectable product (e.g., β-galactosidase, luciferase), or a protein that is itself detectable, e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.; and / or provide a multimerization domain, e.g., a multimerization domain such as the Fc portion of an immunoglobulin. Fusions may also include affinity domains, including peptide sequences that can interact with binding partners, e.g., immobilized on a solid support, useful for identification or purification. Contiguous stretches of a single amino acid, such as histidine, when fused to a protein, are used for one-step purification of the fusion protein by high affinity binding to a resin column such as nickel sepharose.Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO: 58), Hisx6 (HHHHHH) (SEQ ID NO: 59), C-myc (EQKLISEEDL) (SEQ ID NO: 60), Flag (DYKDDDDK) (SEQ ID NO: 61), StrepTag (WSHPQFEK) (SEQ ID NO: 62), hemagglutinin, e.g., HA tag (YPYDVPDYA; SEQ ID NO: 63), glutathione-S-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO: 66), Phe-His-His-Thr (SEQ ID NO: 64), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, W EAAAREACCRECCARA (SEQ ID NO: 65), metal binding domains, e.g., calcium binding proteins such as those from calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, visinin, VILIP, neurocalcin, hippocalcin, frequenin, caltractin, calpain large subunit, S100 proteins, parvalbumin, calbindin D9K, calbindin D28K, and calretinin, zinc binding domains or calcium binding domains, intein, biotin, streptavidin, MyoD, leucine zipper sequences, and maltose binding protein.

[0184] Method for producing humanized anti-factor Bb antibodies In some embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce more desirable (e.g., fully human antibodies) or more robust immune responses can also be used to generate humanized or human antibodies. Examples of such technologies are the Xenomouse® from Amgen, Inc. (Fremont, CA), and the HuMAb-Mouse® and TC Mouse® from Medarex, Inc. (Princeton, NJ), or the H2L2 mouse from Harbour Antibodies BV (Holland). In another alternative, antibodies can be produced recombinantly by phage display or yeast technology. See, e.g., U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al. (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, phage display technology (McCafferty et al. (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

[0185] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by routine methods. For example, F(ab')2 fragments can be produced by pepsin digestion of antibody molecules, and Fab fragments can be produced by reducing the disulfide bridges of F(ab')2 fragments. Genetically engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced, for example, by conventional recombinant techniques. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of a monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into at least one expression vector, which can then be transfected into host cells, such as Escherichia coli (E. coli) cells, monkey COS cells, Chinese hamster ovary (CHO) cells, human HEK293 cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. See, for example, International Publication No. WO 87 / 04462. The DNA can then be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81:6851), or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this way, genetically engineered antibodies, such as "chimeric" or "hybrid" antibodies, with the binding specificity of the target antigen can be prepared.

[0186] Single-chain antibodies can be prepared recombinantly, for example, by linking a nucleotide sequence encoding a heavy chain variable region and a nucleotide sequence encoding a light chain variable region, and in some embodiments, a flexible linker is incorporated between the two variable regions.

[0187] Alternatively, techniques described for the production of single-chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce, for example, phage or yeast scFv libraries, and scFv clones specific for factor Bb can be identified from the libraries according to routine procedures. Positive clones can be subjected to further screening to identify those with high factor Bb binding affinity.

[0188] In some embodiments, humanized anti-factor Bb antibodies are prepared by recombinant techniques as exemplified below. The nucleic acids encoding the heavy and light chains of the anti-factor Bb antibodies described herein can be cloned into one expression vector, with each nucleotide sequence operably linked to an appropriate promoter. In one example, the nucleotide sequences encoding the heavy and light chains are each operably linked to a separate promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains are operably linked to a single promoter, thereby allowing both the heavy and light chains to be expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

[0189] In some cases, the nucleotide sequences encoding the two chains of an antibody can be cloned into two vectors, which can be introduced into the same or different cells. If the two chains are expressed in different cells, each of them can be isolated from the host cell that expresses them, and the isolated heavy and light chains can be mixed and incubated under appropriate conditions to allow antibody formation.

[0190] Generally, the nucleic acid sequence encoding one or all chains of antibody can be operably linked with a suitable promoter and cloned into a suitable expression vector using methods known in the art.For example, the nucleotide sequence and the vector can be contacted under appropriate conditions using a restriction enzyme that creates complementary ends on each molecule that can be paired with each other, and can be joined together using ligase.Alternatively, synthetic nucleic acid linkers can be ligated to the end of the gene.These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector.The choice of expression vector / promoter will depend on the type of host cell used to produce antibody.

[0191] A variety of promoters may be used to express the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate-early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, the E. coli lac UV promoter, and the herpes simplex tk virus promoter.

[0192] Regulatable promoters may also be used, including those that use the lac repressor from Escherichia coli as a transcriptional modulator to regulate transcription from mammalian cell promoters containing the lac operator [Brown, M. et al., Cell, 49:603-612 (1987)] or the tetracycline repressor (tetR) [Gossen, M. and Bujard, H., Proc. Natl. Acad. Sci. USA, 89:5547-555115 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P. et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16 or p65 using estradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad, among others.

[0193] A regulatable promoter containing a repressor can be used in conjunction with an operon. In one embodiment, the lac repressor from Escherichia coli can function as a transcriptional modulator to regulate transcription from a mammalian cell promoter with a lac operator [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)]. The tetracycline repressor (tetR) is combined with a transcriptional activator (VP16) to create the tetR-mammalian cell transcriptional activator fusion protein tTa (tetR-VP16), which is combined with a minimal promoter with tetO derived from the human cytomegalovirus (hCMV) promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When a tetracycline operator is properly placed downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone, rather than a tetR-mammalian cell transcription factor fusion derivative, can function as a potent trans-modulator to regulate gene expression in mammalian cells (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins, which may be toxic to cells in some cases, to achieve its regulatable effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0194] Additionally, the vector may contain some or all of the following: a selectable marker gene, such as a neomycin gene, for the selection of stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high-level transcription; a transcription termination and RNA processing signal from SV40 for mRNA stability; an SV40 polyoma replication origin and ColE1 for proper episomal replication; an internal ribosome binding site (IRES), a versatile multicloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Suitable vectors and methods for producing vectors containing transgenes are well known and available in the art. Examples of polyadenylation signals useful for practicing the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.

[0195] At least one vector (e.g., an expression vector) containing a nucleic acid encoding any of the antibodies can be introduced into a suitable host cell for producing the antibody. The host cell can be cultured under appropriate conditions for expression of the antibody or any of its polypeptide chains. Such antibodies or their polypeptide chains can be recovered by cultured cells (e.g., from the cells or culture supernatant) by conventional methods, such as affinity purification. If necessary, the antibody polypeptide chains can be incubated under appropriate conditions for an appropriate period of time to allow production of the antibody.

[0196] In some embodiments, the method for preparing the antibodies described herein involves a recombinant expression vector encoding both the heavy and light chains of an anti-factor Bb antibody, as also described herein. The recombinant expression vector can be introduced into suitable host cells (e.g., dihydrofolate reductase (DHFR)-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positive transformant host cells can be selected and cultured under appropriate conditions to allow expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium. If necessary, the two chains recovered from the host cells can be incubated under appropriate conditions to allow antibody formation.

[0197] In some embodiments, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-factor Bb antibody and the other encoding the light chain of an anti-factor Bb antibody. Both recombinant expression vectors can be introduced into suitable host cells (e.g., DHFR-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection.

[0198] Alternatively, each expression vector can be introduced into a suitable host cell. Positive transformants can be selected and cultured under appropriate conditions that allow the expression of the antibody polypeptide chains. When two expression vectors are introduced into the same host cell, the antibody produced therein can be recovered from the host cell or culture medium. If necessary, the polypeptide chains can be recovered from the host cell or culture medium, and then incubated under appropriate conditions that allow the formation of antibodies. When two expression vectors are introduced into different host cells, each of them can be recovered from the corresponding host cell or culture medium. The two polypeptide chains can then be incubated under appropriate conditions for the formation of antibodies.

[0199] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography using a matrix coupled to Protein A or Protein G.

[0200] Any of the nucleic acids encoding the heavy chain, light chain, or both of the anti-factor Bb antibodies described herein (e.g., provided in Tables 2 and 3), vectors containing such (e.g., expression vectors); and host cells containing the vectors are within the scope of this disclosure.

[0201] Pharmaceutical Compositions and Treatment Methods Another aspect of the present disclosure provides compositions, including pharmaceutical compositions, comprising any one of the humanized anti-factor Bb antibodies described herein. Generally, pharmaceutical compositions, also referred to herein as formulations, contain an effective amount of any one of the humanized anti-factor Bb antibodies described herein. By "effective amount" is meant a dosage sufficient to bring about a desired result, such as a reduction in adverse symptoms associated with a complement-mediated disease or disorder, an improvement in symptoms of a complement-mediated disease or disorder, or a slowing of progression of a complement-mediated disease or disorder. Generally, the desired result is at least a reduction in symptoms of a complement-mediated disease or disorder compared to a control.

[0202] In the methods of the present disclosure, a humanized anti-factor Bb antibody can be administered to a subject using conventional means capable of producing the desired therapeutic or diagnostic effect. Thus, the humanized anti-factor Bb antibody can be incorporated into a variety of formulations for therapeutic administration. More particularly, the humanized anti-factor Bb antibody can be formulated into a pharmaceutical composition by combining it with a suitable pharmaceutically acceptable carrier, pharmaceutically acceptable diluent, or other pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises a humanized anti-factor Bb antibody and a pharmaceutically acceptable excipient.

[0203] In pharmaceutical dosage forms, the humanized anti-factor Bb antibodies may be administered in the form of their pharmaceutically acceptable salts, or they may be used alone or in appropriate association and combination with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and in no way limiting.

[0204] For oral preparations, the humanized anti-factor Bb antibodies, alone or in combination with suitable excipients, can be used to make tablets, powders, granules, or capsules.

[0205] The humanized anti-factor Bb antibody can be formulated into a preparation for injection by dissolving, suspending, or emulsifying the antibody in an aqueous or non-aqueous solvent; if desired, together with conventional additives such as solubilizing agents, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.

[0206] Pharmaceutical compositions containing humanized anti-factor Bb antibodies are prepared by mixing a humanized anti-factor Bb antibody having the desired purity with optional physiologically acceptable carriers, other excipients, stabilizers, surfactants, buffers, and / or isotonicity agents. Acceptable carriers, other excipients, and / or stabilizers are non-toxic to recipients at the dosages and concentrations employed. In some embodiments, the compositions include buffers, antioxidants, amino acids, or combinations thereof.

[0207] Pharmaceutical compositions can be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, and lyophilized preparations should be reconstituted with sterile solution before administration.The standard procedure for reconstituting lyophilized compositions is to add back a volume of purified water (typically equivalent to the volume removed during lyophilization); however, solutions containing antibacterial agents can be used to produce pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0208] Tonicity agents may be included in the antibody formulation to modulate the tonicity of the formulation. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be appropriate. The term "isotonic" refers to a solution that has the same tonicity as some other solution to which it is compared, such as physiological salt solution or serum.

[0209] Surfactants may also be added to the antibody formulation to reduce aggregation of the formulated antibody and / or minimize particulate formation in the formulation and / or reduce adsorption.

[0210] Lyoprotectants may also be added to protect labile active ingredients (eg, proteins) from destabilizing conditions during the lyophilization process.

[0211] In some embodiments, the subject formulations comprise a humanized anti-factor Bb antibody and at least one of the above-identified agents (e.g., surfactant, buffer, stabilizer, tonicity agent), and are essentially free of at least one preservative.

[0212] Humanized anti-factor Bb antibodies may be utilized in aerosol formulations to be administered by inhalation. Humanized anti-factor Bb antibodies may be formulated into pressurized acceptable propellants.

[0213] Furthermore, the humanized anti-factor Bb antibody can be made into a suppository by mixing it with a variety of bases, such as emulsifying bases or water-soluble bases. The humanized anti-factor Bb antibody can be administered rectally via a suppository.

[0214] Other modes of administration may also find use with the methods of the present disclosure. For example, humanized anti-factor Bb antibodies may be formulated into suppositories, and in some embodiments, into aerosol and intranasal compositions. For suppositories, the vehicle composition may include traditional binders and carriers.

[0215] Intranasal formulations will typically contain a vehicle that does not irritate the nasal mucosa or significantly interfere with ciliary function. Diluents such as water, aqueous saline, or other known substances may be employed. Nasal formulations may also contain a preservative. A surfactant may be present to enhance absorption of the humanized anti-factor Bb antibody by the nasal mucosa.

[0216] Humanized anti-Bb factor antibody can be administered as an injectable preparation.Usually, injectable compositions are prepared as liquid solution or suspension; also prepared as solid form suitable for dissolving or suspending in liquid vehicle before injection.Preparation can also be emulsified, or antibody can be encapsulated in liposome vehicle.

[0217] In some embodiments, the humanized anti-factor Bb antibody is formulated in a controlled release formulation. Controlled release within the scope of this disclosure can be taken to mean any one of several sustained release dosage forms.

[0218] The humanized anti-factor Bb antibody is administered to a subject using any method and route appropriate for drug delivery, including in vivo and ex vivo methods, and systemic and localized routes of administration.

[0219] Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, intranasal, intramuscular, intratracheal, intrathecal, intracranial, subcutaneous, intradermal, topical, intravenous, intraperitoneal, intra-arterial (e.g., via the carotid artery), spinal or cerebral delivery, rectal, nasal, oral, and other enteral and parenteral routes of administration.

[0220] The antibodies of the present disclosure can be administered to a subject using any available conventional method and route suitable for conventional drug delivery, including systemic or localized routes. Generally, routes of administration contemplated by the present disclosure include, but are not necessarily limited to, enteral, parenteral, or inhalation routes.

[0221] In some embodiments, the humanized anti-factor Bb antibody is administered by injection and / or delivery, for example, to a site in a cerebral artery or directly to brain tissue. The humanized anti-factor Bb antibody can also be administered directly to the target site, for example, by biolistic delivery to the target site.

[0222] A variety of subjects can be treated according to the methods provided herein. Generally, such subjects are "mammals" or "mammalian animals," and these terms are used broadly to describe organisms that fall into the class Mammalia, including carnivores (e.g., cats), herbivores (e.g., cows, horses, and sheep), omnivores (e.g., dogs, goats, and pigs), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys). In some embodiments, the subject has a complement system, such as a mammal, fish, or invertebrate. In some embodiments, the subject is a mammal, fish, or invertebrate companion animal, agricultural animal, working animal, zoo animal, or laboratory animal that contains a complement system. In some embodiments, the subject is a human.

[0223] "Treatment" refers to at least an amelioration of symptoms associated with a condition afflicting a subject, where amelioration is used broadly to refer to at least a decrease in the magnitude of a parameter, e.g., a symptom, associated with the condition being treated, such as a complement-mediated disease or disorder. As such, treatment also includes situations in which the condition, or at least the symptoms and / or secondary effects associated therewith, are completely inhibited, e.g., prevented from occurring, or halted, e.g., terminated, such that the subject is no longer afflicted by the condition, or at least the symptoms that characterize the condition.

[0224] In some embodiments, a "subject" is a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), etc. Any animal that has a complement system, such as mammals, fish, and some invertebrates, is also encompassed by these terms. Thus, these terms include companion, agricultural, work, zoo, and laboratory mammals, fish, and invertebrate animals that contain a complement system.

[0225] The term "biological sample" encompasses a variety of sample types obtained from a subject and used in diagnostic or monitoring assays. The definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components such as polynucleotides. The term "biological sample" encompasses clinical samples and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, and / or blood fractions such as plasma and serum. The term "biological sample" also encompasses solid tissue samples, tissue culture samples, and cell samples.

[0226] The present disclosure provides a device or container suitable for containing a composition comprising a humanized anti-factor Bb antibody for administration to a subject. For example, the humanized anti-factor Bb antibody can be placed in a container suitable for containing a pharmaceutical composition. The container can be, for example, a bottle (e.g., with a closure such as a cap), a blister pack (e.g., can provide at least one enclosed dose per blister), a vial, flexible packaging (e.g., a sealed Mylar or plastic bag), an ampoule (for a single dose in solution), a dropper, a syringe, a thin film, and / or a tube. In some embodiments, a container such as a sterile container contains the subject pharmaceutical composition. In some embodiments, the container is a bottle or a syringe. In some embodiments, the container is a bottle. In some embodiments, the container is a syringe. In some embodiments, the device is an injection device such as a syringe (e.g., a pre-filled syringe), a pen (e.g., a pre-filled pen), or an electronic injection device (e-device).

[0227] The present disclosure provides methods for treating complement-mediated diseases or disorders. The methods generally involve administering an effective amount of a humanized anti-factor Bb antibody of the present disclosure to a subject in need thereof. In some embodiments, administration of the humanized anti-factor Bb antibody modulates complement pathway activity in the subject's cells, tissues, or bodily fluids, treating the complement-mediated disease or disorder.

[0228] An "effective amount" refers to the amount of an anti-complement factor Bb antibody that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. A "therapeutically effective amount" will vary depending on the anti-complement factor Bb antibody, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0229] In some embodiments, an effective amount of a humanized anti-factor Bb antibody of the present disclosure is an amount that is effective to reduce or inhibit complement pathway activity in cells, tissues, or bodily fluids of a subject.

[0230] In some embodiments, an effective amount of a humanized anti-factor Bb antibody of the present disclosure is an amount that is effective to reduce or inhibit the formation of MAC in cells, tissues, or bodily fluids of a subject.

[0231] In some embodiments, an effective amount of a humanized anti-factor Bb antibody of the present disclosure is an amount that is effective to reduce or inhibit C3b / Bb-mediated cleavage of C3 in cells, tissues, or bodily fluids of a subject.

[0232] In some embodiments, an effective amount of a humanized anti-factor Bb antibody of the present disclosure is an amount that is effective to reduce or inhibit C3b / Bb-mediated cleavage of C3, thereby reducing the production of C3 cleavage products.

[0233] In some embodiments, an effective amount of a humanized anti-factor Bb antibody of the present disclosure is an amount that is effective to reduce or inhibit complement AP-mediated lysis of cells in a subject.

[0234] In some embodiments, an effective amount of a humanized anti-factor Bb antibody of the present disclosure is an amount that is effective to reduce or inhibit complement AP-mediated hemolysis in a subject's cells, tissues, or bodily fluids (e.g., RBC-containing bodily fluids).

[0235] In some embodiments, an effective amount of a humanized anti-factor Bb antibody of the present disclosure is an amount that is effective to reduce or inhibit the production of anaphylatoxins.

[0236] In some embodiments, an effective amount of a humanized anti-factor Bb antibody of the present disclosure is an amount that is effective to reduce or inhibit AP-mediated deposition of C3b, C3d, or other C3 degradation products on cells or tissues in a subject.

[0237] In some embodiments, an effective amount of a humanized anti-factor Bb antibody of the present disclosure is an amount that is effective to reduce or inhibit AP-mediated C3b deposition on cells or tissues in a subject.

[0238] In some embodiments, an effective amount of a humanized anti-factor Bb antibody of the present disclosure is an amount that is effective to reduce or inhibit AP-mediated deposition of C3b, C3d, or other C3 degradation products on RBCs in a subject.

[0239] In some embodiments, an effective amount of a humanized anti-factor Bb antibody of the present disclosure is an amount that is effective to reduce or inhibit AP-mediated C3b deposition on RBCs in a subject.

[0240] In some embodiments, a humanized anti-factor Bb antibody of the present disclosure, when administered in at least one dose to a subject in need thereof, reduces the amount of circulating factor Bb in the subject. For example, a humanized anti-factor Bb antibody of the present disclosure, when administered in at least one dose to a subject in need thereof, may reduce the amount of circulating factor Bb in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to the amount of circulating factor Bb in the subject in the absence of administration of the humanized anti-factor Bb antibody, or compared to the amount of circulating factor Bb in the subject before administration of the humanized anti-factor Bb antibody.

[0241] In some embodiments, a humanized anti-factor Bb antibody of the present disclosure, when administered in at least one dose to a subject in need thereof, reduces the amount of factor Bb in the subject's plasma. For example, a humanized anti-factor Bb antibody of the present disclosure, when administered in at least one dose to a subject in need thereof, may reduce the amount of factor Bb in the subject's plasma by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to the amount of factor Bb in the subject's plasma in the absence of administration of the humanized anti-factor Bb antibody, or compared to the amount of factor Bb in the subject's plasma before administration of the humanized anti-factor Bb antibody.

[0242] In some embodiments, a method of the present disclosure for treating a subject having a complement-mediated disease or disorder comprises administering to the subject a humanized anti-factor Bb antibody of the present disclosure, or a pharmaceutical composition comprising: a) a humanized anti-factor Bb antibody of the present disclosure; and b) a pharmaceutically acceptable excipient suitable for administration to such a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. Administration can be by any route known to those of skill in the art, including those disclosed herein. In some embodiments, administration is intravenous. In some embodiments, administration is intrathecal. In some embodiments, administration is intramuscular. In some embodiments, administration is subcutaneous.

[0243] Complement-mediated diseases and disorders that are suitable for treatment with the humanized anti-factor Bb antibodies of the present disclosure include diseases and disorders associated with the alternative complement pathway. Investigations in preclinical animal models and clinical trials indicate that alternative pathways play an important role in the development of tissue injury and the pathogenesis of several disease states (Holers et al., Immunological Reviews 223:300-316; Cao et al., (2016) Haematologica 101(11):1319-1326; Schubart et al., (2019) PNAS 116(16):7926-7931; Thurman, (2015) Am J Kidney Dis 65(1):156-168; Vriese et al., (2015) Am J Kidney Dis 65(1):156-168; and Gold et al., (2006) Nat. Genet. 38(4):458-462). In some embodiments, complement-mediated diseases suitable for treatment with a humanized anti-factor Bb antibody of the present disclosure include, but are not limited to, IgA nephropathy (Berge's disease), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), lupus nephritis, ANCA vasculitis, membranous nephropathy, C3 glomerulonephritis (C3GN), focal segmental glomerulosclerosis (FSGS), multiple sclerosis, macular degeneration, age-related macular degeneration (AMD), rheumatoid arthritis, antiphospholipid syndrome, asthma, ischemia-reperfusion injury, membranoproliferative GN type II, spontaneous abortion, microimmune vasculitis, epidermolysis bullosa, recurrent abortion, and traumatic brain injury.

[0244] The humanized anti-factor Bb antibodies of the present disclosure can be administered to a subject in need thereof alone (eg, as monotherapy); or in combination therapy with at least one additional therapeutic agent.

[0245] As used herein, " in combination with " refers to, for example, the first compound is administered during the entire administration period of the second compound; the first compound is administered during the overlapping period with the administration of the second compound, for example, the administration of the first compound begins before the administration of the second compound, and the administration of the first compound ends before the administration of the second compound; the administration of the second compound begins before the administration of the first compound, and the administration of the first compound ends before the administration of the second compound; the administration of the first compound begins before the administration of the second compound, and the administration of the second compound ends before the administration of the first compound; the administration of the first compound begins before the administration of the second compound, and the administration of the second compound ends before the administration of the first compound; the administration of the second compound begins before the administration of the first compound, and the administration of the first compound ends before the administration of the second compound.Therefore, " in combination " can also refer to the regimen that involves the administration of two or more compounds. As used herein, "in combination with" also refers to the administration of two or more compounds administered by the same or different routes, in the same or different formulations, and in the same or different dosage form types.

[0246] Subjects suitable for treatment with a humanized anti-factor Bb antibody include subjects who have been diagnosed with a complement-mediated disease or disorder; subjects who are at greater risk than the general population of developing a complement-mediated disease or disorder (e.g., subjects who have a genetic predisposition to developing a complement-mediated disease or disorder). Also included are subjects with any one of the complement-mediated diseases or disorders listed above. In some embodiments, the subject is an adult. In some embodiments, the subject is a human child. [Example]

[0247] Humanization of a murine monoclonal anti-factor Bb antibody The variable region genes from the parental anti-factor Bb antibody (see Table 1) hybridoma were amplified, cloned and sequenced, resulting in the identification of a single unique VH domain and a single unique Vκ domain.

[0248] First, three humanized VH regions and five humanized Vκ regions designed using COMPOSITE HUMAN ANTIBODY™ technology were cloned into IgG4v1 heavy chain and kappa light chain vectors. The parental antibody, two control antibodies, and all 15 humanized antibody combinations were transiently expressed in HEK EBNA cells.

[0249] To assess the binding of all humanized variants, single-cycle kinetic analysis was performed on supernatants from transfected cell cultures. Kinetic experiments were performed on a Biacore T200 (serial number 1909913) running Biacore T200 Control software V2.0.1 and Evaluation software V3.0 (GE Healthcare, Uppsala, Sweden). All single-cycle kinetic experiments were run at 25°C using HBS-P+ running buffer (pH 7.4) (GE Healthcare, Little Chalfont, UK).

[0250] The antibody was diluted in running buffer to a final concentration of 1 μg / ml based on the concentration assessed by ELISA titer. At the start of each cycle, the antibody was loaded onto Fc2, Fc3, and Fc4 of a Protein A chip (GE Healthcare, Little Chalfont, UK). IgG was captured at a flow rate of 10 μl / min to give an immobilization level (RL) of approximately 63 RU, a theoretical value for achieving an Rmax of approximately 50 RU. The surface was then allowed to stabilize. To minimize any potential mass transport limitations, single-cycle kinetic data were acquired using Bb factor (CompTech, Tyler, USA) as the analyte at a flow rate of 30 μl / min. Multiple repeats using a reference chimeric antibody were performed to check the stability of the surface and analyte over the kinetic cycles. The signal from the reference channel Fc1 (no antibody) was subtracted from that of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. A four-point, two-fold dilution range of factor Bb was used, from 0.78 nM to 6.25 nM, with no regeneration between concentrations. The association phase for four injections of increasing concentrations of factor Bb was monitored for 200 s each, and the dissociation phase was measured once for 200 s after the final injection of factor Bb. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCl, pH 1.5.

[0251] The sensorgrams and fitted data for single-cycle kinetics are shown in Figures 1A-1D, and the kinetic parameters measured for the interaction of factor Bb with each antibody are shown in Table 7. K for VH0 / Vκ0 reference antibody D The relative K is calculated by dividing the K by that of the humanized variant assayed in the same experiment. D was calculated.

[0252] [Table 7]

[0253] Biacore analysis showed that all humanized variants bound to factor Bb; however, in all cases, the relative K D showed a greater than two-fold difference in binding affinity over the chimera, suggesting that some binding affinity was lost. To address this, four additional heavy chain (VH4-VH7) and two light chain (Vκ6-Vκ7) sequences were designed and cloned into appropriate expression vectors. The variant sequences are shown in Tables 2 and 3.

[0254] Expression and single-cycle kinetic analysis of redesigned variants Five control antibodies (VH0 / Vκ6, VH0 / Vκ7, VH5 / Vκ0, VH6 / Vκ0, VH7 / Vκ0) and humanized heavy and light chain combinations (eight total humanized pairings, Table 8) were transiently transfected into HEK EBNA adherent cells (ATCC® Catalog No. CRL-10852™) using the PEI transfection method. IgG supernatant titers were monitored by IgG ELISA (Table 9), and transfections were cultured for up to 10 days, after which supernatants were harvested.

[0255] [Table 8]

[0256] Single-cycle kinetics using cell culture supernatants was performed as described above. Sensorgrams and fitted data for single-cycle kinetics are shown in Figures 2A-2C. All variants were shown to bind to factor Bb. Single-cycle kinetic data (Table 9) demonstrated that five antibodies (VH4 / Vκ6, VH4 / Vκ7, VH6 / Vκ6, VH6 / Vκ7, and VH7 / Vκ7) bound to factor Bb within approximately two-fold of the reference chimeric antibody. The K of the VH0 / Vκ0 reference antibody was approximately 0.01. D The relative K is calculated by dividing the K by that of the humanized variant assayed in the same experiment. D was calculated.

[0257] [Table 9]

[0258] Antibody purification VH4 / Vκ6, VH4 / Vκ7, VH6 / Vκ6, VH6 / Vκ7, and VH7 / Vκ7, as well as chimeric antibodies, were purified from cell culture supernatants on a Protein A Sepharose column (GE Healthcare, Little Chalfont, UK), followed by size exclusion chromatography using a 16 / 60 Superdex 200 column (GE Healthcare, Little Chalfont, UK) with PBS pH 7.4 as the mobile phase. Antibodies were analyzed by OD using extinction coefficients based on the predicted amino acid sequences. 280nm The reduced antibodies were analyzed using SDS-PAGE by loading 1 μg of each antibody onto the gel (FIG. 3), and bands corresponding to a typical antibody profile were observed.

[0259] Multi-cycle kinetic analysis of antibodies To verify the precise affinity for factor Bb, multicycle kinetic analysis was performed on the purified chimeric antibody and five lead antibodies using a Biacore T200 (serial number 1909913) instrument running Biacore T200 Evaluation software V3.0.1 (Uppsala, Sweden). Antibodies were diluted in running buffer to a final concentration of 0.5 μg / ml. At the start of each cycle, antibodies were loaded onto Fc2, Fc3, and Fc4 of a Protein A chip (GE Healthcare, Little Chalfont, UK). IgG was captured at a flow rate of 10 μl / min to give an immobilization level (RL) of approximately 63 RU, a theoretical value for obtaining an Rmax of approximately 50 RU. The surface was then allowed to settle. To minimize any potential mass transport effects, kinetic data were acquired using factor Bb as the analyte at a flow rate of 30 μl / min. Multiple blank and single-concentration analyte repeats were programmed into the kinetic run to check the stability of both the surface and analyte over the kinetic cycles. A two-fold dilution range from 12.5 nM to 0.391 nM Bb factor was selected for kinetic analysis. The association phase of Bb factor was monitored for 360 s, and the dissociation phase was monitored for 600 s. Regeneration of the Protein A surface was performed using two injections of 10 mM glycine-HCl pH 1.5. The signal from the reference channel Fc1 was subtracted from that of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface, and the overall Rmax parameter was used in a one-to-one binding model.

[0260] Sensorgrams and fitted data for binding of the chimeric antibody and humanized variants to factor Bb are shown in Figures 4A-4C. D The relative K is calculated by dividing the K by that of the chimeric antibody on the same chip. D The kinetic parameters measured for the interaction of factor Bb with the chimeric antibody and humanized variants are shown in Table 10. Two humanized variants, VH4 / Vκ6 and VH4 / Vκ7 (bold), had relative K values ​​within 2-fold of the reference chimeric antibody.D showed.

[0261] [Table 10]

[0262] Bb factor competition ELISA The lead purified variant and chimeric antibodies were tested for their binding to factor Bb using competition against the murine parent antibody.

[0263] Factor Bb was diluted to 1.0 μg / ml in 1x PBS and 100 μl / well was coated onto a 96-well ELISA plate overnight at 4°C. The next day, the plate was blocked with 1% casein / PBS for 2 hours at room temperature and then washed twice with PBS pH 7.4. In a 96-well dilution plate, a fixed concentration of murine parent antibody (0.5 μg / ml final concentration) was added in equal volumes to a 4-fold titration series of test antibody diluted in blocking buffer (starting at 45 μg / ml and ending at 0.01 μg / ml final concentration). After washing the plate three times with PBS-T, 100 μl of the chimeric / test antibody mix was added to the ELISA plate. After incubation at room temperature for 1 hour, the plate was washed three times with PBS-T, and bound mouse antibody was detected by applying 100 μl of anti-mouse IgG Fc-specific HRP (Sigma, Dorset, UK) diluted 1:1000 in PBS-T for 1 hour at room temperature. For color development, the plate was washed three times with PBS-T, after which 100 μl of TMB substrate was added and incubated at room temperature for approximately 5 minutes. The reaction was stopped with 50 μl of 3.0 M hydrochloric acid, and the absorbance was immediately read at 450 nm using a DYNEX® plate reader.

[0264] The results are plotted and shown in Figure 5. IC50 values ​​were calculated for each variant, and relative IC50 values ​​were calculated by dividing the IC50 of the humanized variant by that of the chimeric antibody assayed on the same plate (Table 11). All lead variants demonstrated IC50 values ​​within 2-fold of the parent antibody.

[0265] [Table 11] [Example]

[0266] Activity of humanized variants Inhibition of WIESLAB® AP by a humanized variant of anti-Bb factor in human serum The ability of the humanized variants to inhibit complement AP activity was measured using the Complement System Alternative Pathway WIESLAB® kit. In this plate-based assay, lipopolysaccharide (LPS)-coated wells specifically led to activation of the alternative pathway, and detection of membrane attack complex (MAC) deposition served as the readout. 5.56% normal human serum (NHS) was incubated with a dilution series of the parental antibody and humanized variants, along with a human IgG4 control antibody starting at 100 μg / mL. OD405nm was measured and compared with kit positive and negative controls. Data were plotted against the plate positive control (Figure 6A). All humanized variants showed similar inhibition of AP-mediated MAC deposition in human serum as the parental antibody (Table 13).

[0267] [Table 12]

[0268] Inhibition of AP-mediated hemolysis by humanized variants in human serum Inhibition of AP pathway-mediated hemolysis was determined using human or cynomolgus monkey serum and rabbit erythrocytes in a buffer containing EGTA, which inhibits the classical pathway. A dilution series of the parent antibody and humanized variants starting at 200 μg / mL was run in 20% human serum and 10×10 6The mutants were incubated with 100 rabbit red blood cells (RBCs) for 1 hour at 37°C. The amount of lysis was determined by measuring the absorbance of the supernatant at 540 nm and subtracting the background absorbance in control wells containing ethylenediaminetetraacetic acid (EDTA). The results are shown in Figure 6B. In Figure 6C, the A540, representing the amount of hemolysis, is shown for each variant at 100 μg / mL. VH4 / VK6 and VH6 / VK7 showed the greatest inhibition of hemolysis, reflected by the greatest decrease in A540.

[0269] Binding of parental antibodies and humanized variants to cynomolgus monkey factor Bb To ensure that the humanization process does not affect species cross-reactivity, the parent antibody and humanized derivatives were tested for binding to human and cynomolgus monkey factor Bb by biolayer interferometry (BLI) using Octet Red. Briefly, biotinylated antibodies were loaded onto SA biosensors equilibrated in PBS, 0.1% BSA, and 0.02% Tween-20 (assay buffer). After a 60-second baseline in assay buffer, the antibody was loaded onto the probe for 180 seconds, followed by another 60-second baseline. Association to human (Comptech) or cynomolgus monkey factor Bb (purified in-house) was measured for 300 seconds, followed by a 300-second dissociation. Kinetic parameters were calculated using Octet Analysis software using a 1:1 binding model. The data are summarized in Table 13 and demonstrate that humanization does not affect cross-reactivity to cynomolgus monkey factor Bb.

[0270] [Table 13]

[0271] V to human and cynomolgus monkey factor Bb H 4 / V K 6-IgG4v2 and V H 6 / V K 7-IgG4v2 binding To determine whether modifications to the Fc portion of an antibody affect affinity and species cross-reactivity, V H 4 / V K 6-IgG4v2 and V H 6 / V K 7-IgG4v2, respectively, and their parent antibodies V H 4 / V K 6 and V H 6 / V K In parallel with IgG7, VH4 / VK6-IgG4v2 and VH6 / VK7-IgG4v2 were tested by BLI for their ability to bind to human and cynomolgus monkey factor Bb. VH4 / VK6-IgG4v2 and VH6 / VK7-IgG4v2 contained mutations in the Fc region that increased affinity for Fc receptors. The experiment was performed as described in the Examples above, and the results are summarized in Table 14. As expected, the Fc modifications did not affect binding.

[0272] [Table 14]

[0273] V for factor Bb (the activated form of factor B) H 4 / V K 6 and V H 6 / V K 7 Specificities V binds to zymogens factor B and factor Bb from both humans and cynomolgus monkeys H 4 / V K 6 and V H 6 / V KThe capacity of 7 was determined by surface plasmon resonance using a Biacore T200. Human factor B and factor Bb were purchased from Comptech, and cynomolgus factor B and factor Bb were purified in-house. Briefly, monoclonal antibodies were captured on a Biacore Series S Protein A chip in HBSP+ (10 mM HEPES, 150 mM NaCl, 0.05% P20 pH 7.4) at a flow rate of 30 μL / min. A five-point concentration series of each analyte was tested for binding using single-cycle kinetics, with a contact time of 180 seconds per concentration, followed by a dissociation time of 600 seconds at 25°C and a flow rate of 60 μL / sec. Both human and cynomolgus factor B started at a concentration of 500 nM, followed by two-fold dilutions, while human and cynomolgus factor Bb started at concentrations of 30 nM and 150 nM, respectively, followed by two-fold dilutions. The data were analyzed using Biacore evaluation software using a 1:1 binding model. Sensorgrams are shown in Figures 7A-7D. The results are summarized in Table 15. Briefly, both humanized variants showed approximately 10-fold higher affinity for human factor Bb compared to cynomolgus factor Bb. Binding to human or cynomolgus factor B was nearly undetectable, and the small signal observed was dominated by nonspecific binding.

[0274] [Table 15]

[0275] Binding of the chimeric parent and representative humanized variants to various complement proteins To ensure that humanization did not introduce nonspecific binding or cross-reactivity to other complement or plasma proteins, the chimeric parent antibody and a representative humanized variant, VH6 / VK7-IgG4v2, were tested for binding to aC1s (Comptech A104), C1r (Comptech A102), C2 (Comptech A112), C2a (prepared from C2), thrombin (EMD Millipore 605195), elastase (EMD Millipore 324682), Factor D (Comptech A136), and Factor Bb (Comptech A155). Briefly, complement and plasma proteins were coated onto ELISA plates at 2.5 μg / mL in PBS overnight at 4°C. Plates were then blocked with casein for 1 hour at room temperature and washed four times with 1x DPBS / 0.05% Tween-20, followed by a single wash with 1x DPBS. Serial dilutions starting at 50 μg / ml of biotinylated chimeric parent or VH6 / VK7-IgG4v2 were added to the plate in PBS / 0.1% casein / 0.1% Tween-20 and incubated at room temperature for 2 hours. The plate was washed as described, and a 1:10,000 dilution of streptavidin-HRP (Southern Biotech 7100-05) in PBS / 0.1% casein / 0.1% Tween-20 was added to the plate and incubated at room temperature for 30 minutes. The plate was washed again, and Ultra TMP ELISA (Thermo 34028) was added to the plate for 1 minute, followed by stop solution. OD450nm was read on a plate reader, and background at 620nm was subtracted. Both the chimeric parent (Figure 8A) and the humanized variant (Figure 8B) showed complete specificity for factor Bb. [Example]

[0276] Production V from CHO vs HEK H 6 / V K 7-IgG4v2 behaves similarly V produced in HEK cells H 6 / V KTo determine whether 7-IgG4v2 behaves similarly when produced in CHO cells, V7-IgG4v2 was expressed either by transient expression in HEK cells or by stable expression in CHO cells using standard methods. H 6 / V K 7-IgG4v2 was produced.

[0277] The complement alternative pathway (CAP) and classical pathway (CCP) activities of the antibodies were determined using the Complement System Alternative Pathway and Classical Pathway WIESLAB® kits, respectively, according to the manufacturer's instructions. To determine CAP activity, the antibodies were tested in 5.56% normal human serum (FIG. 9A) and 5.56% normal cynomolgus monkey serum (FIG. 9B). To determine CCP activity, the antibodies were tested in 1% normal human serum (FIG. 10A) and 1% normal cynomolgus monkey serum (FIG. 10B). OD 405nm The V produced in CHO and HEK was measured and the results were normalized to the serum activity before antibody injection. H 6 / V K 7-IgG4v2 showed similar activity. IC for both sets of antibodies 50 The values ​​were calculated and are shown in Figures 10A-10B. H 6 / V K 7-IgG4v2 is a V-IgG produced in HEK H 6 / V K 7-IgG4v2. V produced in CHO and HEK showed similar efficacy. H 6 / V K AP pathway-mediated hemolysis of 7-IgG4v2 was also compared. Hemolysis was determined using 20% ​​normal human serum and rabbit red blood cells in a buffer containing EGTA, which inhibits the classical pathway. The amount of lysis was determined by measuring the supernatant and subtracting the background absorbance in control wells containing ethylenediaminetetraacetic acid (EDTA). V produced in CHO and HEK was significantly higher in IgG4v2 than in IgG4v2. H 6 / V K 7-IgG4v2 showed a similar % hemolysis (Figure 11).

[0278] V produced in CHO and HEK H 6 / V K Multicycle kinetic analysis was performed on the 7-IgG4v2 antibody using biolayer interferometry. The antibody was diluted to 10 μg / mL in PBS + 0.02% Tween 20, 0.1% BSA, 0.05% sodium azide and loaded onto an anti-hIgG Fc sensor (pre-equilibrated in the same buffer) for 90 seconds, followed by a 60-second baseline in buffer. Binding of a concentration series of human Factor Bb (Complement Technologies, #A155), ranging from 100 nM to 0.14 nM in two-fold dilutions, was measured in the same buffer for a 300-second association step, followed by a 300-second dissociation step. Binding curves and fitted data for both antibodies, as well as the calculated K d is shown in FIG.

[0279] References Cao et al. (2016) Haematologica 101(11):1319-1326 Chothia et al. (1987) J. Mol. Biol. 196:901-917 Bryson et al. (2010). Biodrugs 24(1):1-8 Dall'Acqua et al. (2006) J Biol Chem 281:23514-24 De Vriese et al. (2015) J Am Soc Nephrol 26:2917-2929 Holers (2008) Immunological Reviews 223:300-316 Holt et al. (2003) Trends Biotechnol. 21:484 Gold et al. (2006) Nat. Genet. 38(4):458-462 Kabat et al., J. Biol. Chem. 252:6609-6616 (1977) Kabat et al. USDept. of Health and Human Services, “Sequences of proteins of immunological interest” (1991) Lefranc et al. (2003) Developmental and Comparative Immunology 27:55 pages MacCallum et al. (1996) J. Mol. Biol. 262:732-745 Perry et al. (2008) Drugs RD 9(6):385-396 Schubart et al. (2019) PNAS 116(16):7926-7931 Smith P et al. (2012) PNAS 109:6181-6186 Shields et al. (2001) J Biol Chem 276:6591-604 Shaner et al. (2005) Nat. Methods 2:905-909 Thurman(2015)Am J Kidney Dis 65(1):156-168 U.S. Patent No. 6,737,056 International Application No. WO 02 / 060919 International Application No. WO 98 / 23289 International Application No. WO 97 / 34631

[0280] All publications, patents, patent applications, publications, and database entries (e.g., sequence database entries) mentioned herein, for example, in the Background, Summary, Detailed Description, Examples, and / or References sections, are hereby incorporated by reference in their entirety, as if each individual publication, patent, patent application, publication, and database entry was specifically and individually incorporated by reference herein. In case of conflict, the present application, including any definitions herein, will control.

[0281] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0282] Articles such as "a," "an," and "the" can mean at least one unless indicated otherwise or otherwise clear from the context. A claim or description including "or" between two or more members of a group is considered to stand when one, more than one, or all of the group members are present, unless indicated otherwise or otherwise clear from the context. The disclosure of a group including "or" between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one member of the group is present, and embodiments in which all of the group members are present. For brevity, such embodiments are not individually detailed herein, but it will be understood that each of these embodiments is provided herein and specifically claimed or disclaimed.

[0283] The present disclosure should be understood to encompass all variations, combinations, and permutations in which at least one limitation, element, clause, or descriptive term from at least one claim or from at least one relevant portion of this description is introduced into another claim. For example, a claim that is dependent on another claim may be amended to include at least one limitation found in any other claim that is dependent on the same base claim. Furthermore, if a claim recites a composition, it should be understood to include methods, if any, of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, unless otherwise indicated or unless it is obvious to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0284] When elements are presented as a list, for example, in Markush group format, it should be understood that any possible subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps. In general, when an embodiment, product, or method is referred to as comprising particular elements, components, or steps, it should be understood that embodiments, products, or methods consisting of or consisting essentially of such elements, components, or steps are also provided. For brevity, such embodiments are not individually detailed herein, but it will be understood that each of these embodiments is provided herein and specifically claimed or disclaimed.

[0285] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "having," "consisting of," and the like, are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the Manual of Patent Examining Procedures, Section 2111.03.

[0286] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and / or the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can, in some embodiments, take on any specific value within the described range, to the nearest tenth of the lower limit of the range, unless the context clearly states otherwise. For simplicity, the values ​​within each range are not individually detailed herein, but it will be understood that each of these values ​​is provided herein and specifically claimed or disclaimed. It should also be understood that, unless otherwise indicated or otherwise apparent from the context and / or the understanding of one of ordinary skill in the art, values ​​expressed as ranges can take on any subrange within the given range, and that the endpoints of the subrange are expressed to the same precision as the nearest tenth of the lower limit of the range.

[0287] When a website is provided, the URL addresses are provided as browser-inoperable code, with the periods of each web address in parentheses. The actual web address does not contain parentheses.

[0288] In addition, it should be understood that any particular embodiment of the present disclosure is expressly excluded from any one or more of the claims. Where a range is provided, any value within the range is expressly excluded from any one or more of the claims. Any embodiment, element, configuration, application, or aspect of the compositions and / or methods of the present disclosure is excluded from any one or more of the claims. For brevity, not all of the embodiments in which at least one element, configuration, purpose, or aspect is excluded are explicitly described herein.

[0289] The terms "about" and "substantially" preceding a numerical value mean ±10% of the recited numerical value.

Claims

1. A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 19, which specifically binds to human complement factor Bb protein. H ) and a light chain variable region (V L ). A humanized antibody comprising:

2. A heavy chain variable region (V) comprising the amino acid sequence of SEQ ID NO: 17, which specifically binds to human complement factor Bb protein. H ) and a light chain variable region (V L ). A humanized antibody comprising:

3. 10 -6 ~10 -9 3. The humanized antibody of claim 1 or claim 2, which specifically binds to human complement factor Bb protein with an affinity of M.

4. The humanized antibody of any one of claims 1 to 3, which inhibits complement pathway activity.

5. The humanized antibody of claim 4, wherein the complement pathway activity is selected from the group consisting of AP-mediated terminal membrane attack complex (MAC) deposition, AP-mediated hemolysis, C3 fragment deposition on red blood cells or other cell types, C3b / Bb-mediated cleavage of C3, and C3bBb3b-mediated cleavage of C5.

6. The humanized antibody of any one of claims 1 to 5, which is a bispecific or multispecific antibody.

7. Ig monomer, Fab fragment, F(ab') 2 The humanized antibody of any one of claims 1 to 6, which is selected from the group consisting of a fragment, scFv, scAb, and Fv.

8. The humanized antibody of any one of claims 1 to 6, comprising a heavy chain constant region of isotype IgG1, IgG2, IgG3, or IgG4.

9. comprising an IgG4 constant region or a variant thereof; The humanized antibody of claim 8, wherein the variant is an IgG4 constant region containing (i) an amino acid sequence having at least one and at most 20 amino acid variations compared to SEQ ID NO: 28, or (ii) an amino acid sequence having at most 20 amino acid variations compared to SEQ ID NO: 29 or 30.

10. The humanized antibody of claim 8, wherein the heavy chain constant region comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 28 to 30.

11. A humanized antibody according to any one of claims 1 or 3 to 10, comprising a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 32 to 34 and a light chain comprising the amino acid sequence of SEQ ID NO:

35.

12. A humanized antibody according to any one of claims 2 to 10, comprising a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 36 to 38 and a light chain comprising the amino acid sequence of SEQ ID NO:

39.

13. A conjugate comprising the humanized antibody of any one of claims 1 to 12.

14. A pharmaceutical composition comprising the humanized antibody of any one of claims 1 to 12 or the conjugate of claim 13.

15. 15. The pharmaceutical composition of claim 14, further comprising a pharmaceutically acceptable excipient.

16. A device comprising the humanized antibody of any one of claims 1 to 12, the conjugate of claim 13, or the pharmaceutical composition of claim 14 or claim 15.

17. 17. The device of claim 16, which is an injection device.

18. The device of claim 17, wherein the injection device is a syringe, a pen, or an electronic injection device (e-device).

19. 16. The pharmaceutical composition of claim 14 or 15 for treating a subject with a complement-mediated disease or disorder.

20. 20. The pharmaceutical composition of claim 19, wherein the complement-mediated disease or disorder is selected from the group consisting of IgA nephropathy (Berge's disease), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), lupus nephritis, ANCA vasculitis, membranous nephropathy, C3 glomerulonephritis (C3GN), focal segmental glomerulosclerosis (FSGS), multiple sclerosis, macular degeneration, age-related macular degeneration (AMD), rheumatoid arthritis, antiphospholipid antibody syndrome, asthma, ischemia-reperfusion injury, type II membranoproliferative GN, spontaneous abortion, microimmune vasculitis, epidermolysis bullosa, recurrent abortion, and traumatic brain injury.

21. 16. The pharmaceutical composition of claim 14 or 15 for use in a method of inhibiting complement pathway activity in a subject.

22. 22. The pharmaceutical composition of claim 21, wherein the subject has a complement-mediated disease or disorder.

23. Complement-mediated diseases or disorders include IgA nephropathy (Berge's disease), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), lupus nephritis, ANCA vasculitis, membranous nephropathy, 23. The pharmaceutical composition of claim 22, wherein the disease is selected from the group consisting of glaucoma, C3 glomerulonephritis (C3GN), focal segmental glomerulosclerosis (FSGS), multiple sclerosis, macular degeneration, age-related macular degeneration (AMD), rheumatoid arthritis, antiphospholipid syndrome, asthma, ischemia-reperfusion injury, type II membranoproliferative GN, spontaneous abortion, microimmune vasculitis, epidermolysis bullosa, recurrent abortion, and traumatic brain injury.

24. 24. The pharmaceutical composition of any one of claims 21 to 23, wherein the complement pathway activity is selected from the group consisting of AP-mediated terminal membrane attack complex (MAC) deposition, AP-mediated hemolysis, C3 fragment deposition on red blood cells or other cell types, C3b / Bb-mediated cleavage of C3, and C3bBb3b-mediated cleavage of C5.

25. The pharmaceutical composition according to any one of claims 19 to 24, wherein the pharmaceutical composition is for intravenous, subcutaneous, or intramuscular administration.

26. A nucleic acid or set of nucleic acids that encodes or collectively encodes the humanized antibody of any one of claims 1 to 12.

27. A vector or vector set comprising the nucleic acid or nucleic acid set of claim 26.

28. A cell expressing the humanized antibody of any one of claims 1 to 12, the nucleic acid or nucleic acid set of claim 26, or the vector or vector set of claim 27.

29. 29. The cell of claim 28, which is a mammalian cell.

30. 30. The cell of claim 29, wherein the mammalian cell is selected from the group consisting of a human embryonic kidney (HEK) cell, a Chinese hamster ovary (CHO) cell, an NSO myeloma cell, an SP2 cell, a COS cell, and a mammary epithelial cell.

31. A method for producing a humanized antibody, comprising the step of culturing the cell according to any one of claims 28 to 30 to produce the humanized antibody.

32. 32. The method of claim 31, further comprising isolating the humanized antibody.

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