Humanized anti-complement factor Bb antibody and its use

Abstract

The present invention provides a humanized anti-Bb factor antibody, a composition containing the antibody, a method for producing the antibody, and a method for using the antibody, for treating complement-mediated diseases or disorders. [Solution] A humanized anti-Bb factor antibody is provided that combines only a specific heavy chain variable region (VH) and light chain variable region (VL) to bind to Bb factor with an acceptable binding affinity. The humanized anti-Bb factor antibody of this disclosure has a binding affinity of no more than twice that of the parent antibody.

Description

[Technical Field]

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

[0002] This application relates to a humanized anti-Bb factor antibody and the use of the antibody. [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 phagocytic cells to remove harmful pathogens from an organism. The complement system comprises three distinct upstream activation pathways (classical, alternative, and lectin pathways), all converging on a common terminal pathway. Factor B is a component of the alternative pathway of the complement system and is cleaved into factors Ba and Bb. Factor B also contains a serine protease (SP) domain, which, when activated, provides catalytic activity for 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, from autoimmune diseases to organ transplantation. There remains a need to treat diseases or disorders related to the complement system. This invention addresses this need and others. [Overview of the Initiative] [Means for solving the problem]

[0004] This disclosure provides, in certain embodiments, humanized anti-Bb factor antibodies, compositions comprising such antibodies, methods for producing such antibodies, and methods for using such antibodies, for example, to treat complement-mediated diseases or disorders. As shown in the data provided herein, the humanized anti-Bb factor antibodies of this disclosure have a binding affinity of no more than twice that of the parental antibody. Unexpectedly, initial attempts to humanize parental mouse anti-Bb factor antibodies produced a majority of variants lacking acceptable binding affinity. Thus, multiple rounds of humanization were required to produce a humanized version with appropriate binding affinity (for example, to treat complement-mediated diseases or disorders). Furthermore, certain V H and V L By combining only the domains, we were able to produce antibodies that bind to the Bb factor with acceptable binding affinity; see, for example, the antibodies tested in Example 1, Table 9.

[0005] Some aspects of this disclosure relate to a heavy chain variable region (V) that specifically binds to human complement factor Bb protein and includes the amino acid sequence of SEQ ID NO: 19. H ) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 27 L The present invention provides humanized antibodies containing )

[0006] Other aspects of this disclosure relate to a V protein that specifically binds to human complement factor Bb protein and includes the amino acid sequence of SEQ ID NO: 17. H and V containing the amino acid sequence of SEQ ID NO: 26 L This provides humanized antibodies containing [specific antibodies].

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

[0008] In some embodiments, humanized antibodies inhibit complement pathway activity. In some embodiments, complement pathway activity is alternative pathway (AP) activity.

[0009] In some embodiments, 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 humanized antibody is a bispecific antibody or a multispecific antibody.

[0010] In some embodiments, the humanized antibody is selected from the group consisting of Ig monomer, Fab fragment, F(ab’)2 fragment, scFv, scAb, and Fv.

[0011] In some embodiments, the humanized antibody comprises a heavy chain constant region of isotype IgG1, IgG2, IgG3, or IgG4.

[0012] In some embodiments, the humanized antibody comprises an IgG4 constant region or a variant thereof.

[0013] 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.

[0014] In some embodiments, the humanized antibody comprises a heavy chain comprising any one of the amino acid sequences of SEQ ID NOs: 32-34 and a light chain comprising the amino acid sequence of SEQ ID NO: 35.

[0015] In some embodiments, the humanized antibody comprises a heavy chain comprising any one of the amino acid sequences of SEQ ID NOs: 36-38 and a light chain comprising the amino acid sequence of SEQ ID NO: 39.

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

[0017] Conjugates comprising the humanized antibodies of the present disclosure are also provided herein.

[0018] In some embodiments, the humanized anti-Bb factor antibody of the conjugate comprises a V comprising the amino acid sequence of SEQ ID NO: 19 Hand V containing the amino acid sequence of SEQ ID NO: 27 L This includes. In some embodiments, the conjugate humanized anti-Bb factor antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 34 and a light chain containing the amino acid sequence of SEQ ID NO: 35.

[0019] In some embodiments, the conjugate humanized anti-Bb factor antibody contains the amino acid sequence V of SEQ ID NO: 17. H and V containing the amino acid sequence of SEQ ID NO: 26 L This includes. In some embodiments, the conjugate humanized anti-Bb factor antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 38 and a light chain containing the amino acid sequence of SEQ ID NO: 39.

[0020] Pharmaceutical compositions comprising a humanized antibody or a conjugate as described herein are further provided herein.

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

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

[0023] In some embodiments, the pharmaceutical composition contains the amino acid sequence of SEQ ID NO: 19. H and V containing the amino acid sequence of SEQ ID NO: 27 L Conjugate containing humanized anti-Bb factor antibodies The compound includes a conjugate comprising a humanized anti-Bb factor 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 contains the amino acid sequence V of SEQ ID NO: 17. H and V containing the amino acid sequence of SEQ ID NO: 26 L The pharmaceutical composition includes a conjugate containing a humanized anti-Bb factor antibody. In some embodiments, the pharmaceutical composition includes a conjugate containing a humanized anti-Bb factor antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 38 and a light chain containing the amino acid sequence of SEQ ID NO: 39.

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

[0026] Devices comprising humanized antibodies, conjugates, or pharmaceutical compositions described herein are also provided herein.

[0027] In some embodiments, the device includes the amino acid sequence V of sequence number 19. H and V containing the amino acid sequence of SEQ ID NO: 27 L The device includes a humanized anti-Bb factor antibody. In some embodiments, the device includes a humanized anti-Bb factor antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 34 and a light chain containing the amino acid sequence of SEQ ID NO: 35.

[0028] In some embodiments, the device includes the amino acid sequence V of SEQ ID NO: 17. H and V containing the amino acid sequence of SEQ ID NO: 26 L The device includes a humanized anti-Bb factor antibody. In some embodiments, the device includes a humanized anti-Bb factor antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 38 and a light chain containing the amino acid sequence of SEQ ID NO: 39.

[0029] In some embodiments, the device includes the amino acid sequence V of sequence number 19. H and V containing the amino acid sequence of SEQ ID NO: 27 L The device includes a conjugate containing a humanized anti-Bb factor antibody. In some embodiments, the device includes a conjugate containing a humanized anti-Bb factor antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 34 and a light chain containing the amino acid sequence of SEQ ID NO: 35.

[0030] In some embodiments, the device includes the amino acid sequence V of SEQ ID NO: 17. H and V containing the amino acid sequence of SEQ ID NO: 26 L The device includes a conjugate containing a humanized anti-Bb factor antibody. In some embodiments, the device includes a conjugate containing a humanized anti-Bb factor antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 38 and a light chain containing the amino acid sequence of SEQ ID NO: 39.

[0031] In some embodiments, the device includes the amino acid sequence V of sequence number 19. H and V containing the amino acid sequence of SEQ ID NO: 27 L The device comprises a pharmaceutical composition comprising a humanized anti-Bb factor antibody. In some embodiments, the device comprises a pharmaceutical composition comprising a humanized anti-Bb factor antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 34 and a light chain containing the amino acid sequence of SEQ ID NO: 35.

[0032] In some embodiments, the device includes the amino acid sequence V of SEQ ID NO: 17. H and V containing the amino acid sequence of SEQ ID NO: 26 L The device comprises a pharmaceutical composition comprising a humanized anti-Bb factor antibody. In some embodiments, the device comprises a pharmaceutical composition comprising a humanized anti-Bb factor antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 38 and a light chain containing 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, and It is an electronic injection device (e-device).

[0034] Further aspects of this disclosure provide a method for treating a subject having a complement-mediated disorder, comprising the step of administering an effective amount of a humanized antibody, a conjugate, or a pharmaceutical composition described herein to the subject to treat the complement-mediated disorder.

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

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

[0037] In some embodiments, a method for treating a subject with a complement-mediated disease or disorder comprises the amino acid sequence of SEQ ID NO: 19. H and V containing the amino acid sequence of SEQ ID NO: 27 LThe method includes administering an effective amount of a conjugate containing a humanized anti-Bb factor antibody to a subject to treat a complement-mediated disease or disorder. In some embodiments, the method for treating a subject having a complement-mediated disease or disorder includes administering an effective amount of a conjugate containing a heavy chain containing the amino acid sequence of SEQ ID NO: 34 and a light chain containing the amino acid sequence of SEQ ID NO: 35 to a subject to treat a complement-mediated disease or disorder.

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

[0039] In some embodiments, a method for treating a subject with a complement-mediated disease or disorder comprises the amino acid sequence of SEQ ID NO: 19. H and V containing the amino acid sequence of SEQ ID NO: 27 L The method includes administering an effective amount of a pharmaceutical composition containing a humanized anti-Bb factor antibody to a subject to treat a complement-mediated disease or disorder. In some embodiments, the method for treating a subject having a complement-mediated disease or disorder includes administering an effective amount of a pharmaceutical composition containing a heavy chain containing the amino acid sequence of SEQ ID NO: 34 and a light chain containing the amino acid sequence of SEQ ID NO: 35 to a subject to treat a complement-mediated disease or disorder.

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

[0041] In some embodiments, complement-mediated diseases are selected from the group consisting of IgA nephropathy (Berger'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] Further embodiments of this disclosure provide methods for inhibiting complement pathway activity in a subject. In some embodiments, complement pathway activity is alternative pathway (AP) activity. In some embodiments, a method for inhibiting complement pathway activity (e.g., AP activity) in a subject includes administering an effective amount of a humanized anti-Bb factor antibody, a conjugate containing a humanized anti-Bb factor antibody, or a pharmaceutical composition containing a humanized anti-Bb factor antibody to the subject to inhibit complement pathway activity.

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

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

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

[0046] In some embodiments, a method for inhibiting complement pathway activity (e.g., AP activity) in a target includes the amino acid sequence of SEQ ID NO: 17. H and V containing the amino acid sequence of SEQ ID NO: 26 LThe method includes administering an effective amount of a conjugate containing a humanized anti-Bb factor antibody to a target to inhibit complement pathway activity. In some embodiments, the method for inhibiting complement pathway activity (e.g., AP activity) in a target involves administering a conjugate containing a humanized anti-Bb factor antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 38 and a light chain containing the amino acid sequence of SEQ ID NO: 39. The process includes administering an effective dose of the substance to inhibit complement pathway activity.

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

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

[0049] In some embodiments, complement AP activity is selected from the group consisting of AP-mediated terminal membrane invasion complex (MAC) deposition, AP-mediated hemolysis, C3 fragment deposition on erythrocytes or other cell types, C3b / Bb-mediated cleavage of C3, and C3b / Bb / 3b-mediated cleavage of C5. In some embodiments, the subject has a complement-mediated disease or disorder.

[0050] In some embodiments, the method further includes the step of administering a therapeutic agent to a target.

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

[0052] Humanized anti-Bb factor antibodies for use in methods for treating complement-mediated diseases or disorders are also provided herein. Conjugates comprising humanized anti-Bb factor antibodies for use in methods for treating complement-mediated diseases or disorders are further provided herein. Pharmaceutical compositions comprising humanized anti-Bb factor antibodies for use in methods for treating complement-mediated diseases or disorders are further provided herein. Devices comprising humanized anti-Bb factor antibodies for use in methods for treating complement-mediated diseases or disorders are further provided herein.

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

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

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

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

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

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

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

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

[0061] In some embodiments, complement-mediated diseases are selected from the group consisting of IgA nephropathy (Berger'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 gnosis, spontaneous abortion, microimmunovasculitis, epidermolysis bullosa, recurrent abortion, and traumatic brain injury.

[0062] Humanized anti-Bb factor antibodies for use in methods for inhibiting complement pathway activity (e.g., AP activity) are also provided herein. A conjugate comprising a humanized anti-Bb factor antibody for use in methods for inhibiting complement pathway activity (e.g., AP activity) is provided herein. Further details are provided herein. Pharmaceutical compositions comprising humanized anti-Bb factor antibodies for use in methods for inhibiting complement pathway activity (e.g., AP activity) are further provided herein. Devices comprising humanized anti-Bb factor antibodies for use in methods for inhibiting complement pathway activity (e.g., AP activity) are further provided herein.

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

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

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

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

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

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

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

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

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

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

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

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

[0075] In some embodiments, the vector or vector set comprises a nucleic acid or nucleic acid set encoding or collectively encoding a humanized anti-Bb factor antibody comprising the amino acid sequence of SEQ ID NO: 17 V H and the amino acid sequence of SEQ ID NO: 26 V L In some embodiments, the vector or vector set comprises a nucleic acid or nucleic acid set encoding or collectively encoding a humanized anti-Bb factor 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 cell comprises a humanized anti-Bb factor antibody comprising the amino acid sequence of SEQ ID NO: 19 V H and the amino acid sequence of SEQ ID NO: 27 V L In some embodiments, the cell comprises a humanized anti-Bb factor 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 cell comprises a humanized anti-Bb factor antibody comprising the amino acid sequence of SEQ ID NO: 17 V H and the amino acid sequence of SEQ ID NO: 26 V L In some embodiments the cell comprises a humanized anti-Bb factor 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 cell comprises a nucleic acid or nucleic acid set encoding or collectively encoding a humanized anti-Bb factor antibody comprising the amino acid sequence of SEQ ID NO: 19 V H and the amino acid sequence of SEQ ID NO: 27 V L In some embodiments, the cell comprises a nucleic acid or nucleic acid set encoding or collectively encoding a humanized anti-Bb factor 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 cell contains the amino acid sequence of SEQ ID NO: 17. H and V containing the amino acid sequence of SEQ ID NO: 26 L The cells include nucleic acids or sets of nucleic acids that encode or collectively encode humanized anti-Bb factor antibodies, including the sequence of amino acids of SEQ ID NO: 38 and the sequence of amino acids of SEQ ID NO: 39.

[0080] In some embodiments, the cell contains the amino acid sequence of SEQ ID NO: 19. H and V containing the amino acid sequence of SEQ ID NO: 27 L The vector or vector set comprises nucleic acids or sets of nucleic acids encoding or collectively encoding humanized anti-Bb factor antibodies, including the sequence of amino acids of SEQ ID NO: 34 and the sequence of amino acids of SEQ ID NO: 35.

[0081] In some embodiments, the cell contains the amino acid sequence of SEQ ID NO: 17. H and V containing the amino acid sequence of SEQ ID NO: 26 L The vector or vector set comprises nucleic acids or sets of nucleic acids encoding or collectively encoding humanized anti-Bb factor antibodies, including the sequence of amino acids of SEQ ID NO: 38 and the sequence of amino acids of SEQ ID NO: 39.

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

[0083] Other embodiments of this disclosure provide a method for producing a humanized antibody as described herein, comprising the step of culturing cells as described herein to produce a humanized antibody. In some embodiments, the method further comprises the step of isolating the humanized antibody.

[0084] In some embodiments, a method for producing a humanized anti-Bb factor antibody involves the amino acid sequence of SEQ ID NO: 19. H and V containing the amino acid sequence of SEQ ID NO: 27 L The method for producing humanized anti-Bb factor antibodies includes culturing cells containing nucleic acids or a set of nucleic acids that collectively encode humanized anti-Bb factor antibodies, including the amino acid sequence of SEQ ID NO: 34 and the amino acid sequence of SEQ ID NO: 35.

[0085] In some embodiments, a method for producing a humanized anti-Bb factor antibody involves the amino acid sequence of SEQ ID NO: 17. H and V containing the amino acid sequence of SEQ ID NO: 16 L By culturing cells containing nucleic acids or sets of nucleic acids that collectively encode humanized anti-Bb factor antibodies, The method includes a step of producing a humanized anti-Bb factor antibody. In some embodiments, the method for producing a humanized anti-Bb factor antibody includes a step of culturing cells containing a nucleic acid or a set of nucleic acids that collectively encode a humanized anti-Bb factor antibody, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 28 and a light chain containing the amino acid sequence of SEQ ID NO: 39.

[0086] The above summary is intended to illustrate, in a non-limiting manner, some 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 attached drawings are not intended to be drawn to scale. In the drawings, identical or nearly identical components illustrated in different drawings are represented by similar numbers. For clarity, not all components are labeled in every drawing. [Brief explanation of the drawing]

[0088] [Figure 1A] This graph 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 the binding of humanized variants and control antibodies to factor Bb. Kinetic analysis was performed using a Biacore T200. Each antibody was captured on a Protein A CM5 chip, and then increasing concentrations of factor Bb were injected to determine the single-cycle off-rate. *The scale is the same for each subdivision, with x ranging from -200 to 1400 and y from -5 to 40. [Figure 1B] This graph 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 the binding of humanized variants and control antibodies to factor Bb. Kinetic analysis was performed using a Biacore T200. Each antibody was captured on a Protein A CM5 chip, and then increasing concentrations of factor Bb were injected to determine the single-cycle off-rate. *The scale is the same for each subdivision, with x ranging from -200 to 1400 and y from -5 to 40. [Figure 1C]This graph 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 the binding of humanized variants and control antibodies to factor Bb. Kinetic analysis was performed using a Biacore T200. Each antibody was captured on a Protein A CM5 chip, and then increasing concentrations of factor Bb were injected to determine the single-cycle off-rate. *The scale is the same for each subdivision, with x ranging from -200 to 1400 and y from -5 to 40. [Figure 1D] This graph 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 the binding of humanized variants and control antibodies to factor Bb. Kinetic analysis was performed using a Biacore T200. Each antibody was captured on a Protein A CM5 chip, and then increasing concentrations of factor Bb were injected to determine the single-cycle off-rate. *The scale is the same for each subdivision, with x ranging from -200 to 1400 and y from -5 to 40. [Figure 2A] This graph shows the binding of the humanized variant (group 2) to factor Bb using single-cycle kinetics. Raw sensorgrams and fitted curves (1:1 binding model) for the binding of the redesigned variant and control antibody to factor Bb are shown. Kinetic analysis was performed using a Biacore T200. Each antibody was captured on a Protein A CM5 chip, and then increasing concentrations of factor Bb were injected to determine the single-cycle off-rate. [Figure 2B] This graph shows the binding of the humanized variant (group 2) to factor Bb using single-cycle kinetics. Raw sensorgrams and fitted curves (1:1 binding model) for the binding of the redesigned variant and control antibody to factor Bb are shown. Kinetic analysis was performed using a Biacore T200. Each antibody was captured on a Protein A CM5 chip, and then increasing concentrations of factor Bb were injected to determine the single-cycle off-rate. [Figure 2C]This graph shows the binding of the humanized variant (group 2) to factor Bb using single-cycle kinetics. Raw sensorgrams and fitted curves (1:1 binding model) for the binding of the redesigned variant and control antibody to factor Bb are shown. Kinetic analysis was performed using a Biacore T200. Each antibody was captured on a Protein A CM5 chip, and then increasing concentrations of factor Bb were injected to determine the single-cycle off-rate. [Figure 3] This figure shows SDS-PAGE gels of purified Protein A / Post-SEC antibodies. 1 μg of each reducing antibody sample was loaded onto a NuPage 4-12% Bis-Tris gel (ThermoFisher, Loughborough, UK) and run at 200V for 35 minutes. The gel was stained with Instant Blue (Expedeon, Swavesey, UK). Mk: PAGERuler® Plus pre-stained protein ladder (ThermoFisher, Loughborough, UK). [Figure 4A] This graph shows the binding of lead antibodies (all from group 2) to the Bb factor using multicycle kinetics. Multicycle sensorogram data and fitted curves (1:1 binding model) are shown for humanized variant binding to the Bb factor (Figure 4A: Chimeric VH0 / VK0, VH4 / Vκ6; Figure 4B: VH4 / Vκ7, VH6 / Vκ6; Figure 4C: VH6 / Vκ7, VH7 / Vκ7). [Figure 4B] This graph shows the binding of lead antibodies (all from group 2) to the Bb factor using multicycle kinetics. Multicycle sensorogram data and fitted curves (1:1 binding model) are shown for humanized variant binding to the Bb factor (Figure 4A: Chimeric VH0 / VK0, VH4 / Vκ6; Figure 4B: VH4 / Vκ7, VH6 / Vκ6; Figure 4C: VH6 / Vκ7, VH7 / Vκ7). [Figure 4C]This graph shows the binding of lead antibodies (all from group 2) to the Bb factor using multicycle kinetics. Multicycle sensorogram data and fitted curves (1:1 binding model) are shown for humanized variant binding to the Bb factor (Figure 4A: Chimeric VH0 / VK0, VH4 / Vκ6; Figure 4B: VH4 / Vκ7, VH6 / Vκ6; Figure 4C: VH6 / Vκ7, VH7 / Vκ7). [Figure 5] This graph shows the results of an immunosorbent assay (ELISA) of a humanized variant of Bb factor competitive enzyme against parental antibodies. To assess Bb factor binding, a dilution series of anti-Bb factor variants was tested against fixed-concentration mouse parental antibodies. Binding mouse antibodies were detected using anti-mouse peroxidase conjugate and tetramethylbenzidine (TMB) substrate. [Figure 6A] This graph shows the activity of humanized anti-Bb factor antibodies in the WIESLAB® complement alternative pathway (CAP) (Figure 6A) and AP-mediated hemolysis (Figures 6B-6C) using human serum. [Figure 6B] This graph shows the activity of humanized anti-Bb factor antibodies in the WIESLAB® complement alternative pathway (CAP) (Figure 6A) and AP-mediated hemolysis (Figures 6B-6C) using human serum. [Figure 6C] This graph shows the activity of humanized anti-Bb factor antibodies in the WIESLAB® complement alternative pathway (CAP) (Figure 6A) and AP-mediated hemolysis (Figures 6B-6C) using human serum. [Figure 7A] These graphs show the specificity of human B factor (Figures 7A and 7B) or cynomolgus monkey B factor (Figures 7C and 7D) to their active form (Bb factor) based on surface plasmon resonance. [Figure 7B] These graphs show the specificity of human B factor (Figures 7A and 7B) or cynomolgus monkey B factor (Figures 7C and 7D) to their active form (Bb factor) based on surface plasmon resonance. [Figure 7C]These graphs show the specificity of human B factor (Figures 7A and 7B) or cynomolgus monkey B factor (Figures 7C and 7D) to their active form (Bb factor) based on surface plasmon resonance. [Figure 7D] These graphs show the specificity of human B factor (Figures 7A and 7B) or cynomolgus monkey B factor (Figures 7C and 7D) to their active form (Bb factor) based on surface plasmon resonance. [Figure 8] This graph shows the binding specificity of the chimeric parent antibody VH0 / VK0-IgG4v1 (Figure 8A) and the representative humanized variant antibody VH6 / VK7-IgG4v2 (Figure 8B) to the Bb factor among various complement proteins. [Figure 9] This graph shows the activity of VH6 / VK7-IgG4v2 (produced from HEK cells) and VH6 / VK7-IgG4v2_CHO (produced from CHO cells) in the WIESLAB® complement alternative pathway (CAP) assay using normal human (Figure 9A) and cynomolgus monkey (Cyno) (Figure 9B) serum. [Figure 10] This graph shows 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] This graph shows the hemolysis of rabbit RBCs by VH6 / VK7-IgG4v2_HEK and VH6 / VK7-IgG4v2_CHO in normal human serum. [Figure 12] This graph shows fitted curves representing affinity and multicycle sensor gram data for the binding of VH6 / VK7-IgG4v2_CHO (left) and VH6 / VK7-IgG4v2_HEK (right) to human Bb protein. [Modes for carrying out the invention]

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

[0090] The term "antibody" encompasses any isotype of antibody or immunoglobulin, including but not limited to humanized antibodies and chimeric antibodies. Antibodies can be single-chain antibodies (scAb) or single-domain antibodies (dAb) (e.g., single-domain heavy-chain antibodies or single-domain light-chain antibodies; see Holt et al. (2003) Trends Biotechnol. 21: p. 484). The term "antibody" also encompasses antibody fragments (antibody fragments) that retain specific binding to an antigen. "Antibody" refers to the heavy chain (V) of an antibody linked by a short linker peptide. H ) and light chain (V L A single-chain variable fragment (scFv), which is a fusion protein of the variable region of ), and V linked by a small peptide linker. H and V L This further includes diabodies, which are non-covalent dimers of scFv fragments (Zapata et al., Protein Eng. 8(10): pp. 1057-1062 (1995)). Other fusion proteins, including the antigen-binding portion of an antibody and non-antibody proteins, are also encompassed by the term "antibody."

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

[0092] Papain digestion of antibodies produces two identical antigen-binding fragments, referred to as "Fab" fragments, each having a single antigen-binding site, and the remainder being an "Fc" fragment, a name reflecting its ability to readily crystallize. Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites and can still crosslink to the antigen.

[0093] "Fv" is the minimal antibody fragment containing a complete antigen recognition site and antigen binding site. This region contains a dimer of one heavy chain variable domain and one light chain variable domain in a tightly non-covalent bond. The three CDRs of each variable domain interact to form V H -V L It is this stereochemistry that defines the 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 the Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind to the antigen, albeit with lower affinity than the entire binding site.

[0094] The "Fab" fragment contains a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab fragment differs from the Fab' fragment by the addition of several 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 herein designation for Fab', where the cysteine ​​residue of the constant domain has a free thiol group. The F(ab')2 antibody fragment was originally produced as a pair of Fab' fragments with a hinge cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0095] The "scFv" antibody fragment is the V of the antibody. H and V L These regions are present in a single polypeptide chain. In some cases, the Fv polypeptide is V H and V LThe region further contains polypeptide linkers, which allow the scFv to form the desired structure for antigen binding. For a review of scFv, see Pluckthun in See The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269–315 (1994).

[0096] A "diabody" refers to a small antibody fragment that has two antigen-binding sites, and the fragment is on the same polypeptide chain, V L V connected H (V H -V L ) includes. By using a linker that is too short to pair two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are described more thoroughly, for example, by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: pp. 6444-6448 (1993).

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

[0098] Antibodies can be detected by labeling them with, for example, radioisotopes, enzymes that produce detectable products, and / or fluorescent proteins. Antibodies can be further conjugated to other parts, such as members of specific binding pairs, for example, the biotin member of the biotin-avidin specific binding pair. Antibodies can also be conjugated to solid supports, including but not limited to polystyrene plates and / or beads.

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

[0100] A "monoclonal antibody" is an antibody produced entirely by a group of identical cells, each produced from a single cell through repeated cell replication. In other words, a cell clone produces only a single antibody species. Monoclonal antibodies are produced using hybridoma production techniques, but other production methods known to those skilled in the art may also be used (e.g., antibodies derived from antibody phage display libraries).

[0101] A "complementarity-determining region (CDR)" is a discontinuous antigen-binding site found within the variable regions of both heavy and light chain polypeptides. CDRs were described by Lefranc et al. (2003) in "Developmental and Comparative Immunology." As described in 27:55; 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); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), the definitions include duplication or subsets of amino acid residues when compared to one another. Nevertheless, the application of any definition to refer to the CDR of an antibody or graft antibody or its variant is intended to be within the scope of the terms 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 in the light chain variable region, respectively. As used herein, the terms "CDR-H1," "CDR-H2," and "CDR-H3" refer to the first, second, and third CDRs in the heavy chain variable region, respectively. As used herein, the terms "CDR-1," "CDR-2," and "CDR-3" refer to the first, second, and third CDRs in the variable region of either chain, respectively.

[0103] When used in relation to the antibody variable region, the "framework" includes all amino acid residues outside the CDR region within the antibody's variable region. A variable region framework is generally a discontinuous amino acid sequence containing only those amino acids outside the CDR. The "framework region" includes each domain of the framework separated by the CDR.

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

[0105] In some embodiments, humanized antibodies are produced using synthetic and / or recombinant nucleic acids to prepare a gene (e.g., cDNA) encoding the desired humanized chain. For example, nucleic acid (e.g., DNA) sequences encoding the humanized variable region can be constructed using PCR mutagenesis methods that modify 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., Nucleic 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, a mutation can be introduced into a cloned variable region, and a sequence encoding a variant with desired specificity can be selected (e.g., from a phage library; see, for example, Krebber et al., U.S. Patent No. 5,514,548; and Hoogenboom et al., International Publication No. 93 / 06213, published April 1, 1993).

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

[0107] [Table 1]

[0108] In some embodiments, the humanized anti-Bb factor antibody described herein includes heavy chain complementarity determination region 1 (CDR-H1), heavy chain complementarity determination region 2 (CDR-H2), and heavy chain complementarity determination region 3 (CDR-H3) of the heavy chain variable region containing the amino acid sequence of SEQ ID NO: 12. In some embodiments, the humanized anti-Bb factor antibody described herein includes light chain complementarity determination region 1 (CDR-L1), light chain complementarity determination region 2 (CDR-L2), and light chain complementarity determination region 3 (CDR-L3) of the light chain variable region containing the amino acid sequence of SEQ ID NO: 13. In some embodiments, the humanized anti-Bb factor antibody further includes 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-Bb factor antibody described herein comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 1, CDR-H2 containing the amino acid sequence of SEQ ID NO: 2, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 3. In some embodiments, according to the Kabat definition, the humanized anti-Bb factor antibody described herein comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 4, CDR-L2 containing the amino acid sequence of SEQ ID NO: 5, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the humanized anti-Bb factor antibody further comprises 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-Bb factor antibody described herein comprises CDR-H1 containing the amino acid sequence of SEQ ID NO: 7, CDR-H2 containing the amino acid sequence of SEQ ID NO: 8, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, according to the IMGT definition, the humanized anti-Bb factor antibody described herein comprises CDR-L1 containing the amino acid sequence of SEQ ID NO: 10, CDR-L2 containing the amino acid sequence of SEQ ID NO: 11, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 6. In some embodiments, the humanized anti-Bb factor antibody further comprises a humanized heavy chain framework region and / or a humanized light chain framework region.

[0110] In some embodiments, the humanized anti-Bb factor antibody described herein is V as described in SEQ ID NO: 12. H In comparison, V contains at most 20 amino acid variations (for example, 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 variation). H This includes. In some embodiments, the anti-Bb factor antibody of this disclosure is V as described in SEQ ID NO: 13. L In comparison, V contains at most 20 amino acid variations (for example, at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation). L Includes.

[0111] In some embodiments, the humanized anti-Bb factor antibody described herein is V as described in SEQ ID NO: 12. H V containing an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to H Includes. In some embodiments, the humanized anti-Bb factor of this disclosure is V as described in SEQ ID NO: 13. L V containing 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, the humanized anti-Bb factor antibody of this disclosure comprises CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 1, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 2, CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 3, and V as described in SEQ ID NO: 12. H In comparison, humanized V contains at most 20 amino acid variations in the framework region (for example, 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 variation). HThis includes. In some embodiments, the humanized anti-Bb factor antibody of this disclosure comprises CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 4, CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 6, and V as described in SEQ ID NO: 13. L In comparison, humanized V contains at most 20 amino acid variations in the framework region (for example, 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 variation). L Includes.

[0113] In some embodiments, the humanized anti-Bb factor antibody of this disclosure comprises humanized V-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 1, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 3. H including, V H The framework area is collectively V as described in Sequence ID No. 12. H The framework region is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to that of the present invention. In some embodiments, the humanized anti-Bb factor antibody of the present disclosure includes humanized V, which comprises CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 4, CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 6. L including, V L The framework area is collectively V as described in any one of sequence numbers 13. L It is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the framework domain.

[0114] In some embodiments, the humanized anti-Bb factor antibody of this disclosure comprises CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 7, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 8, CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 9, and V as described in SEQ ID NO: 12. H In comparison, the framework region has at most 20 amino acid variations (for example, many Humanized V contains (20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation), H This includes. In some embodiments, the humanized anti-Bb factor antibody of this disclosure comprises CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 10, CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 11, and CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 6, and V as described in SEQ ID NO: 13. L In comparison, humanized V contains at most 20 amino acid variations in the framework region (for example, 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 variation). L Includes.

[0115] In some embodiments, the humanized anti-Bb factor antibody of this disclosure comprises humanized V, CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 7, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 9. H including, V H The framework area is collectively V as described in Sequence ID No. 12. HThe framework region is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to that of the present invention. In some embodiments, the humanized anti-Bb factor antibody of this disclosure includes humanized V, which comprises CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 10, CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 11, and CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 6. L including, V L The framework area is collectively V as described in any one of sequence numbers 13. L It is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the framework domain.

[0116] Examples of amino acid sequences and DNA coding sequences of the humanized heavy chain variable region of the humanized anti-Bb factor antibodies described herein are provided in Table 2. Examples of amino acid sequences and DNA coding sequences of the humanized light chain variable region of the humanized anti-Bb factor 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, the humanized anti-Bb factor antibody of this disclosure comprises CDR-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 1, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 3, and V as described in any one of SEQ ID NOs. 14 to 20. H In comparison, humanized V contains at most 20 amino acid variations in the framework region (for example, 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 variation). H This includes. In some embodiments, the humanized anti-Bb factor antibody of this disclosure comprises CDR-L1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 4, CDR-L2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 5, and CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 6, and V as described in any one of SEQ ID NOs. 21 to 27. L In comparison, humanized V contains at most 20 amino acid variations in the framework region (for example, 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 variation). L Includes.

[0120] In some embodiments, the humanized anti-Bb factor antibody of this disclosure comprises humanized V-H1 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 1, CDR-H2 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 2, and CDR-H3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 3. H including, V H The framework area is collectively described in one of sequence numbers 14-20. HThe framework region is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the framework region. In some embodiments, the humanized anti-Bb factor antibody of this disclosure is CDR-L1 (according to the Kabat defined system) having the amino acid sequence of SEQ ID NO: 4, CDR-L2 (according to the Kabat defined system) having the amino acid sequence of SEQ ID NO: 5 Humanized V (according to the Kabat definition system), which includes CDR-L3 (according to the Kabat definition system) having the amino acid sequence of SEQ ID NO: 6. L including, V L The framework area is collectively V as described in any one of sequence numbers 21-27. L It is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the framework domain.

[0121] In some embodiments, the humanized anti-Bb factor antibody of this disclosure comprises CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 7, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 8, CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 9, and V described in any one of SEQ ID NOs. 14 to 20. H In comparison, humanized V contains at most 20 amino acid variations in the framework region (for example, 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 variation). H This includes. In some embodiments, the humanized anti-Bb factor antibody of this disclosure comprises CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 10, CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 11, and CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 6, and V as described in any one of SEQ ID NOs. 21 to 27. LIn comparison, humanized V contains at most 20 amino acid variations in the framework region (for example, 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 variation). L Includes.

[0122] In some embodiments, the humanized anti-Bb factor antibody of this disclosure comprises humanized V, CDR-H1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 7, CDR-H2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 8, and CDR-H3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 9. H including, V H The framework area is V, which is described in one of sequence numbers 14-20. H The framework region is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to that of the present invention. In some embodiments, the humanized anti-Bb factor antibody of this disclosure includes humanized V, which comprises CDR-L1 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 10, CDR-L2 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 11, and CDR-L3 (according to the IMGT definition system) having the amino acid sequence of SEQ ID NO: 6. L including, V L The framework area is collectively V as described in any one of sequence numbers 21-27. L It is at least 80% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the framework domain.

[0123] In some embodiments, the humanized anti-Bb factor antibody of this disclosure is the humanized V described in any one of SEQ ID NOs: 14-20. H This includes. In some embodiments, the humanized anti-Bb factor antibody of this disclosure is the humanized V described in any one of SEQ ID NOs. 21 to 27. L Includes. Table 4 shows the humanized V provided in Table 2. H One of the humanized Vs provided in Table 3 LThis provides an example of a humanized anti-Bb factor antibody, including one of the following.

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

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

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

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

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

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

[0130] In some embodiments, the humanized anti-Bb factor antibody of this disclosure is a humanized V antibody containing the amino acid sequence of SEQ ID NO: 14. HHumanized V containing either one of the amino acid sequences of SEQ ID NO: 21 L Includes.

[0131] In some embodiments, the humanized anti-Bb factor antibody of this disclosure is the amino acid of SEQ ID NO: 14 Humanized V containing sequences H Humanized V containing either one of the amino acid sequences of SEQ ID NO: 22 L Includes.

[0132] In some embodiments, the humanized anti-Bb factor antibody of this disclosure is a humanized V antibody containing the amino acid sequence of SEQ ID NO: 14. H Humanized V containing either one of the amino acid sequences of SEQ ID NO: 23 L Includes.

[0133] In some embodiments, the humanized anti-Bb factor antibody of this disclosure is a humanized V antibody containing the amino acid sequence of SEQ ID NO: 14. H Humanized V containing either one of the amino acid sequences of SEQ ID NO: 24 L Includes.

[0134] In some embodiments, the humanized anti-Bb factor antibody of this disclosure is a humanized V antibody containing the amino acid sequence of SEQ ID NO: 14. H Humanized V containing either one of the amino acid sequences of SEQ ID NO: 25 L Includes.

[0135] In some embodiments, the humanized anti-Bb factor antibody described herein is full-length IgG, Ig monomer, Fab fragment, F(ab')2 fragment, scFv, scAb, or Fv. In some embodiments, the humanized anti-Bb factor antibody described herein is full-length IgG. In some embodiments, the heavy chain of any of the humanized anti-Bb factor antibodies described herein includes a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, e.g., human, mouse, rat, or rabbit. In some embodiments, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., 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-Bb factor antibodies described herein. In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) may be introduced into the heavy chain constant region (numbered according to the Kabat numbering system (e.g., the EU index in Kabat), for example, in the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1) and / or the hinge region) to increase or decrease the antibody's affinity for Fc receptors (e.g., activated Fc receptors) on the surface of effector cells. Mutations in the Fc region of antibodies that decrease or increase the affinity of antibodies for Fc receptors, and techniques for introducing such mutations into Fc receptors or fragments thereof, are known to those skilled in the art. Examples of mutations in antibodies at the Fc receptor to alter the affinity of an antibody to the Fc receptor are incorporated herein by reference, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publications 02 / 060919; 98 / 23289; and 97 / 34631.

[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. Altering the number of cysteine ​​residues in the hinge region of the CH1 domain may, for example, promote the association of the light and heavy chains, or alter the stability of the antibody (e.g., increase or decrease it), or promote linker conjugation.

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

[0139] In some embodiments, the constant region described herein is the IgG1 constant region, numbered according to the EU index as in Kabat, and includes 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. See U.S. Patent No. 7,658,921, incorporated herein by reference. This type of mutant IgG, referred to as the "YTE variant," 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 281:235 pp. 14-24). In some embodiments, the antibody includes an IgG constant domain containing 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 EU indices as well as Kabat. Additional mutations introduced into the heavy chain constant region that would increase the antibody half-life are known in the art, for example, the M428L / N434S (EU numbering; M459L / N466S Kabat numbering) mutation described by Zalevsky et al., Nat Biotechnol. February 2010; 28(2): pp. 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 whose affinity is altered may be, for example, the Fc receptor or the C1 component of complement. This technique is described in more detail in U.S. Patents 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation (by point mutation or other means) of the constant domain may reduce the Fc receptor binding of the circulating antibody, thereby increasing tumor localization. For a description of mutations that delete or inactivate the constant domain and thereby increase tumor localization, see, for example, U.S. Patents 5,585,097 and 8,591,886. In some embodiments, at least one amino acid substitution can be introduced into the Fc region of the antibodies described herein to remove a potential glycosylation site on the Fc region that may reduce Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276: pp. 6591-604).

[0141] In some embodiments, an antibody may have at least one amino acid in its constant region replaced with a different amino acid residue so that it has altered Clq binding and / or reduced or absent complement-dependent cell-mediated cytotoxicity (CDC). This technique is described in more detail in U.S. Patent 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 modified to thereby alter the antibody's ability to bind to complement. This technique is described in more detail in International Publication No. 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 cell-mediated cytotoxicity (ADCC) and / or increase the antibody's affinity for the Fcγ receptor. This technique is described in more detail in International Publication No. 00 / 42072.

[0142] In some embodiments, to avoid potential complications resulting from Fab-arm exchange, which is known to occur with native IgG4 mAbs, the antibodies provided herein may include 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, pp. 105-108; 1993). In some embodiments, to reduce residual antibody-dependent cellular cytotoxicity, an L235E (corresponding to L248E in Kabat numbering, EU numbering) mutation is introduced into 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-Bb factor 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, any light chain of the humanized anti-Bb factor antibodies described herein may further include a light chain constant region (C L ). In some examples, C L is a kappa light chain. In other examples, C L is a lambda light chain. In some embodiments, C L is a kappa light chain, and its sequence is provided below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(Sequence ID 31)

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

[0147] In some embodiments, the humanized anti-Bb factor antibodies described herein are V listed in Table 2. H The heavy chain comprises one of the following or any variant thereof, and a heavy chain constant region which is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to one of SEQ ID NOs. 28-30. In some embodiments, the humanized anti-Bb factor antibodies described herein are V listed in Table 2. H The heavy chain comprises one of the following or any variant thereof, and a heavy chain constant region containing at least 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 variation) compared to any one of SEQ ID NOs. In some embodiments, the humanized anti-Bb factor antibodies described herein are V listed in Table 2. H It comprises a heavy chain containing any one of the above or any variant thereof, and a heavy chain constant region containing any one amino acid sequence from SEQ ID NOs. 28 to 30.

[0148] In some embodiments, the humanized anti-Bb factor antibodies described herein are V listed in Table 3. LThe light chain comprises one of the following or any variant thereof, and a light chain constant region which 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-Bb factor antibody described herein is V listed in Table 3. L The light chain comprises one of the following or any variant thereof, and a light chain constant region containing at least 20 amino acid variations compared to SEQ ID NO: 31 (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 variation). In some embodiments, the humanized anti-Bb factor antibody described herein is V listed in Table 3. L A light chain containing one of the above or any variant thereof, and a light chain constant region containing the amino acid sequence of SEQ ID NO: 31.

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

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

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

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

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

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

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

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

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

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

[0159] In some embodiments, the humanized anti-Bb factor antibody of this disclosure is approximately 10 -6 ~10 -11 nM, for example, 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 M affinity. The term "approximately" preceding the numerical value means ±10% of the given value.

[0160] In some embodiments, the humanized anti-Bb factor antibody of the present disclosure exhibits preferential binding to the Bb factor as compared to binding to the B factor. In some embodiments, the humanized anti-Bb factor antibody of the present disclosure binds to the Bb factor but does not substantially bind to the soluble B factor. In some embodiments, the humanized anti-Bb factor antibody of the present disclosure binds to the Bb factor 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 affinity of the antibody for the B factor. In some embodiments, the humanized anti-Bb factor antibody of the present disclosure binds to the Bb factor 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 the B factor factor.

[0161] In some embodiments, the humanized anti-Bb factor antibody of the present disclosure inhibits 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% as compared to the level of complement activity in the absence of the humanized anti-Bb factor antibody.

[0162] In some embodiments, the humanized anti-Bb factor 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% as compared to the level of AP activity in the absence of the humanized anti-Bb factor antibody. In some embodiments, the humanized anti-Bb factor antibody of the present disclosure has an IC -7 M to 10 -9 M of IC50 For example, 10 -7 M~5×10 -7 M, 5×10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5×10 -8 M~10 -9 M IC 50 This inhibits AP activity. In some embodiments, complement AP activity is selected from the group consisting of AP-mediated terminal membrane invasion complex (MAC) deposition, AP-mediated hemolysis, C3 fragment deposition on erythrocytes or other cell types, C3b / Bb-mediated cleavage of C3, and C3b / Bb / 3b-mediated cleavage of C5. In some embodiments, inhibition of complement AP activity by humanized anti-Bb factor antibodies is measured using the Complement System Alternative Pathway WIESLAB® kit.

[0163] In some embodiments, the humanized anti-Bb factor antibody of this disclosure inhibits the formation of membrane invasion complexes (MACs) 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 MACs formed in the absence of the anti-Bb factor antibody.

[0164] In some embodiments, the humanized anti-Bb factor antibody of this disclosure inhibits C3b / Bb-mediated cleavage of C3. C3b / Bb is also known as "C3 convertase". In some embodiments, the humanized anti-Bb factor antibody of this disclosure inhibits 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 C3 cleavage in the absence of the anti-Bb factor antibody. In some embodiments, the humanized anti-Bb factor antibody of this disclosure inhibits C3b / Bb-mediated cleavage of C3 by at least 10%.-7 M~10 -9 M IC 50 For example, 10 -7 M~5×10 -7 M, 5×10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5×10 -8 M~10 -9 M IC 50 This inhibits C3b / Bb-mediated cleavage of C3.

[0165] In some embodiments, the humanized anti-Bb factor antibody of the Disclosure inhibits C3b / Bb-mediated cleavage of C3, thereby reducing the production of C3 cleavage products. For example, the humanized anti-Bb factor antibody of the 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-Bb factor antibody.

[0166] In some embodiments, the humanized anti-Bb factor antibody of this disclosure inhibits 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 degree of cytolysis in the absence of the anti-Bb factor antibody. The degree of inhibition of AP-mediated cytolysis can be determined using a cytolysis assay. In some embodiments, the humanized anti-Bb factor antibody of this disclosure inhibits complement AP-mediated cytolysis by at least 10%. -7 M~10 -9 M IC 50 For example, 10 -7 M~5×10 -7 M, 5×10 -7 M~10 -8M, 10 -8 M~5×10 -8 M, or 5×10 -8 M~10 -9 M IC 50 This inhibits AP-mediated cell lysis.

[0167] In some embodiments, the humanized anti-Bb factor antibody of this disclosure inhibits 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 degree of hemolysis in the absence of the anti-Bb factor antibody. The degree of inhibition of AP-mediated hemolysis can be determined using a rabbit erythrocyte (RBC) hemolysis assay. In some embodiments, the humanized anti-Bb factor antibody of this disclosure inhibits complement AP-mediated hemolysis by at least 10%. -7 M~10 -9 M IC 50 For example, 10 -7 M~5×10 -7 M, 5×10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5×10 -8 M~10 -9 M IC 50 This inhibits AP-mediated hemolysis.

[0168] In some embodiments, the humanized anti-Bb factor antibody of this disclosure inhibits AP-mediated deposition of C3b, C3d, or other C3 degradation products on cells or tissues. For example, the humanized anti-Bb factor antibody of this disclosure may 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 deposition of C3b, C3d, or other C3 degradation products on cells or tissues in the absence of or before administration of the anti-Bb factor antibody. In some embodiments, the humanized anti-Bb factor antibody of this disclosure may 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 deposition of C3b, C3d, or other C3 degradation products on cells or tissues in the absence of or before administration of the anti-Bb factor antibody. -7 M~10 -9 M IC 50 For example, 10 -7 M~5×10 -7 M, 5×10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5×10 -8 M~10 -9 M IC 50 This inhibits AP-mediated deposition of C3b, C3d, or other C3 degradation products on cells or tissues.

[0169] In some embodiments, the humanized anti-Bb factor antibody of the Disclosure inhibits AP-mediated C3b deposition 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 deposition on cells or tissues in the absence of the humanized anti-Bb factor antibody. -7 M~10 -9 M IC 50 For example, 10 -7 M~5×10 -7M, 5×10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5×10 -8 M~10 -9 M IC 50 This inhibits AP-mediated C3b deposition on cells or tissues.

[0170] In some embodiments, the humanized anti-Bb factor antibody of this disclosure reduces AP-mediated C3b deposition on red blood cells (RBCs) or other cell types by at least 10%, at least 20%, compared to the amount of C3b deposition on RBCs in the absence of the humanized anti-Bb factor antibody. It inhibits by 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%. In some embodiments, the humanized anti-Bb factor antibody of this disclosure is 10 -7 M~10 -9 M IC 50 For example, 10 -7 M~5×10 -7 M, 5×10 -7 M~10 -8 M, 10 -8 M~5×10 -8 M, or 5×10 -8 M~10 -9 M IC 50 This inhibits AP-mediated C3b deposition on RBCs.

[0171] In some embodiments, the humanized anti-Bb factor antibody of the Disclosure, when administered to a subject in need, reduces the amount of circulating Bb factor in the subject. For example, when administered to a subject in need, the humanized anti-Bb factor antibody of the Disclosure may reduce the amount of circulating Bb factor 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 Bb factor in the subject in the absence of administration of the humanized anti-Bb factor antibody, or compared to the amount of circulating Bb factor in the subject before administration of the humanized anti-Bb factor antibody.

[0172] In some embodiments, the humanized anti-Bb factor antibody of the Disclosure inhibits C3bBb3b-mediated cleavage of C5. In some embodiments, the humanized anti-Bb factor antibody of the Disclosure inhibits 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-Bb factor antibody.

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

[0174] Immunoconjugate In some embodiments, the humanized anti-Bb factor antibody of this disclosure is conjugated with another active substance to form an immunoconjugate. For example, the humanized anti-Bb factor antibody may contain a free thiol (-SH) group at its carboxyl terminus, and the free thiol group can be used to attach the antibody to a second polypeptide (e.g., another antibody including the humanized anti-Bb factor antibody), a scaffold, a carrier, etc. In some embodiments, the humanized anti-Bb factor antibody contains at least one amino acid that does not exist in nature. In some embodiments, the amino acid that does not exist in nature includes a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group. For suitable amino acids that do not exist in nature, see, for example, U.S. Patent No. 7,632,924. The encapsulation of an amino acid that does not exist in nature may provide conjugation to polymers, second polypeptides, scaffolds, etc. For example, a humanized anti-Bb factor antibody linked to a water-soluble polymer (e.g., PEG) can be produced by reacting an antibody with a water-soluble polymer containing a carbonyl group, and the antibody contains a non-naturally encoded amino acid containing an aminooxy, hydrazine, hydrazide, or semicarbazide group. In some embodiments, a humanized anti-Bb factor antibody linked to a water-soluble polymer can be produced 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 via an amide linkage.

[0175] In some embodiments, the humanized anti-Bb factor antibody is linked to a polymer (e.g., a polymer other than a polypeptide) (e.g., covalently). 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, for example, 5,000 Da to 40,000 Da or 25,000 to 40,000 Da.

[0176] In some embodiments, the humanized anti-Bb factor antibody includes a “radiopaque” label, for example, a label readily visible using X-rays. Radiopaque materials are well known to those skilled in the art. The most common radiopaque materials include iodides, bromides, or barium salts. Other radiopaque materials are also known and include, but are not limited to, organic bismuth derivatives (see, e.g., U.S. Patent No. 5,939,045), radiopaque multiurethanes (see, e.g., U.S. Patent No. 5,346,981), organic bismuth hybrids (see, e.g., U.S. Patent No. 5,256,334), and / or radiopaque barium polymer complexes (see, e.g., U.S. Patent No. 4,866,132).

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

[0178] In some embodiments, humanized anti-Bb factor antibodies are 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, duracyte, reaction tray wells (e.g., multiwell plates), plastic tubes, etc. Solid supports may include 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-Bb factor antibodies on a solid support are well known and include, but are not limited to, ionic, hydrophobic, and / or covalent interactions. Solid supports may be soluble or insoluble in aqueous solutions, for example. In some embodiments, suitable solid supports are generally insoluble in aqueous solutions.

[0179] In some embodiments, the humanized anti-Bb factor antibody includes a detectable label. Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Suitable ones 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), and radiolabels (e.g., 3 H, 125 I, 35 S, 14 C, or 32P) includes, but is not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and others commonly used in enzyme-linked immunosorbent assays (ELISA)), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0180] In some embodiments, the humanized anti-Bb factor antibody comprises a contrast agent or radioisotope, and the contrast agent or radioisotope is used for imaging, for example, imaging performed on humans. It is appropriate for use in the law. Non-exclusive examples of the mark are: 123 I (iodine), 18 F (fluorine), 99 Tc (technetium), 111 In (indium), and 67 It contains radioactive isotopes such as Ga (gallium), as well as contrast agents such as gadolinium (Gd), dysprosium, and iron. Radioactive Gd isotope ( 153Gd) can also be used and is suitable for imaging methods in non-human mammals. Humanized anti-Bb factor antibodies can be labeled using standard techniques. For example, humanized anti-Bb factor antibodies can be iodized using chloramine T or 1,3,4,6-tetrachloro-3α,6α-diphenylglycouryl. For fluorination, fluorine is added to the humanized anti-Bb factor antibody during synthesis by fluoride ion substitution reaction. For reviews on the synthesis of proteins 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-Bb factor antibodies can also be labeled with contrast agents using standard techniques. For example, humanized anti-Bb factor antibodies can be labeled with Gd by conjugating them with a low-molecular-weight Gd chelate such as Gd-diethylenetriaminepentaacetic acid (GdDTPA) or Gd-tetraazacyclododecanetetraacetic acid (GdDOTA). See Caravan et al., Chem. Rev. 99: pp. 2293-2352 (1999) and Lauffer et al., J. Magn. Reson. Imaging, 3: pp. 11-16 (1985). For example, humanized anti-Bb factor antibodies can be labeled with Gd by conjugating them with a polylysine-Gd chelate. See, for example, Curtet et al., Invest. Radio., 33(10): pp. 752-761 (1998). Alternatively, in some embodiments, humanized anti-Bb factor antibodies can be labeled with Gd by incubating paramagnetic polymerized liposomes containing a Gd chelating lipid with avidin and biotinylated antibodies. See, for example, Sipkins et al., Nature Med., 4:623-626 (1998).

[0181] Suitable fluorescent proteins include green fluorescent protein (GFP) or its variants, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant 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 This includes, but is not limited to, GFP, paGFP, maple protein and kindling protein, β-phycoerythrin, R-phycoerythrin, and phycobiliproteins and phycobiliprotein conjugates, including allophycocyanin. 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: pp. 905-909). Any of the diverse fluorescent and colored proteins from anthozoan species, for example, Matz et al. (1999) Nature Biotechnol. 17: pp. 969-973, may be suitable for use.

[0182] In some embodiments, a humanized anti-Bb factor antibody is conjugated to a therapeutic agent. Any of the humanized anti-Bb factor antibodies disclosed herein may be used to form an antibody-active agent conjugate. The active agent may be conjugated at the N-terminus of the light chain, the C-terminus of the light chain, the N-terminus of the heavy chain, and The active agent may be attached to the C-terminus of the heavy chain. In some embodiments, the active agent is attached to the hinge of the antibody or to at least one other site on the antibody. With respect to single-chain antibodies, the active agent may be attached to the N-terminus or C-terminus of the single-chain antibody. The active agent may be conjugated to the antibody directly or via a linker using techniques known to those skilled in the art. The linker may be cleavable or incleavable. 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-Bb factor antibody is ligated to a fusion partner, such as a ligand; epitope tag; peptide; and / or a protein other than the antibody (e.g., covalently or noncovalently). Appropriate fusion partners include peptides and polypeptides that provide enhanced in vivo stability (e.g., enhanced serum half-life); ease of purification, e.g., (His)n, e.g., 6His; secretion of the fusion protein from cells; epitope tags, e.g., GST, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO: 55), FLAGs (e.g., DYKDDDDK; SEQ ID NO: 56), and / or c-myc (e.g., EQKLISEEDL; SEQ ID NO: 57); detectable signals, e.g., enzymes producing detectable products (e.g., β-galactosidase, luciferase), or proteins that are themselves detectable, e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.; and / or multimerization, e.g., multimerization domains such as the Fc portion of immunoglobulins. The fusion may also include affinity domains, including peptide sequences that can interact with the binding partner, such as those immobilized on a solid support, which are useful for identification or purification. When a sequence of single amino acids, such as histidine, is fused to a protein, it can be used for one-step purification of the fusion protein by achieving high affinity binding to resin columns such as nickel Sepharose.Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO: 58), His×6 (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) includes metal-binding domains, such as those derived from calcium-binding proteins, such as calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, bicinin, VILIP, neurocalcin, hypocalcin, phryquenin, caltractin, calpain large subunit, S100 protein, parvalbumin, calbindin D9K, calbindin D28K, and calretinin, as well as zinc-binding domains or calcium-binding domains, intein, biotin, streptavidin, MyoD, leucine zipper sequences, and maltose-binding proteins.

[0184] Method for producing humanized anti-Bb factor antibodies In some embodiments, fully human antibodies can be obtained by using commercially available mice engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or more robust immune response can also be used for humanization or the production of human antibodies. Examples of such techniques include Xenomouse® from Amgen, Inc. (Fremont, CA), and HuMAb-Mouse® and TC Mouse® from Medarex, Inc. (Princeton, NJ), or H2L2 mice from Harbour Antibodies BV (Holland). In alternative methods, antibodies can be synthesized recombinantly by phage display or yeast technology. For example... See, for example, U.S. Patents 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12: pp. 433-455. Alternatively, phage display technology (McCafferty et al., (1990) Nature 348: pp. 552-553) can be used to produce human antibodies and antibody fragments in vitro from the immunoglobulin variable (V) domain gene repertoire 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 crosslinks of the F(ab')2 fragments. Genetically modified antibodies such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies can be produced, for example, by conventional recombination techniques. In one example, DNA encoding a monoclonal antibody specific to a target antigen can be readily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Upon isolation, the DNA can be placed in at least one expression vector, which is then 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. 87 / 04462. Subsequently, the DNA can be modified, for example, by substituting coding sequences for human heavy and light chain constant domains for homologous mouse sequences (Morrison et al., (1984) Proc. Nat. Acad. Sci. 81: 6851), or by covalently conjugating all or part of the coding sequence for non-immunoglobulin polypeptides to the immunoglobulin coding sequence. In this way, genetically modified antibodies, such as "chimeric" or "hybrid" antibodies, with binding specificity to target antigens can be prepared.

[0186] Single-chain antibodies can be prepared by recombinant technology, for example, by linking a nucleotide sequence encoding a heavy-chain variable region with a nucleotide sequence encoding a light-chain variable region. 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. Patents 4,946,778 and 4,704,692) can be adapted to produce, for example, a phage or yeast scFv library, and scFv clones specific to Bb factor can be identified from the library using routine procedures. Positive clones can be subjected to further screening to identify those with high Bb factor binding affinity.

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

[0189] In some cases, the nucleotide sequences encoding two types of antibody chains can be cloned into two vectors and introduced into the same or different cells. If expressed in such a host cell, each of these can be isolated from the host cell expressing such a host cell, and the isolated heavy and light chains can be mixed and incubated under appropriate conditions that allow for antibody formation.

[0190] Generally, nucleic acid sequences encoding one or all of the antibody chains can be cloned into a suitable expression vector by operably ligating them with a suitable promoter using methods known in the art. For example, the nucleotide sequence and vector can be brought into contact under appropriate conditions using restriction enzymes that create complementary ends on each molecule that can pair with each other, and then joined together using ligases. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences corresponding to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell for use in producing the antibody.

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

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

[0193] A moduloable promoter containing a repressor may be used with the operon. In one embodiment, a lac repressor from E. coli can function as a transcription modulator that regulates transcription from mammalian cell promoters 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)]. A tetracycline repressor (tetR) is combined with a transcription activator (VP16) to create a tetR-mammalian cell transcription activator fusion protein tTa (tetR-VP16), which is then combined with a minimal promoter containing tetO, derived from the human cytomegalovirus (hCMV) promoter, to create a tetR-tet operator system that controls gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When a tetracycline operator is properly positioned downstream of the TATA element of the CMVIE promoter, the tetracycline repressor (tetR) alone, rather than the tetR-mammalian cell transcription factor fusion derivative, can function as a potent transmodulator for regulating gene expression in mammalian cells (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline-inducible switch is that it achieves its tunable effect by eliminating the tetracycline, which in some cases can be toxic to cells. The absence of cyclin repressor-mammalian cell transactivators or repressor fusion proteins is a key feature (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, for example: selectable marker genes, such as the neomycin gene, for the selection of stable or transient transfectants in mammalian cells; enhancer / promoter sequences from the earliest genes of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyomatous replication origin and ColE1 for proper episomal replication; internal ribosome binding sites (IRES), multipurpose multicloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Methods for producing suitable vectors and 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, the human collagen I polyadenylation signal, the human collagen II polyadenylation signal, and the SV40 polyadenylation signal.

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

[0196] In some embodiments, the methods for preparing the antibodies described herein involve a recombinant expression vector encoding both the heavy and light chains of an anti-Bb factor 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 suitable conditions to enable the expression of the two polypeptide chains that form the antibody, and these 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 suitable conditions to enable antibody formation.

[0197] In some embodiments, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-Bb factor antibody and the other encoding the light chain of an anti-Bb factor 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 to enable the expression of the antibody polypeptide chain. If two expression vectors are introduced into the same host cell, the antibodies produced therein can be recovered from the host cell or culture medium. If necessary, the polypeptide chain can be recovered from the host cell or culture medium and then incubated under appropriate conditions to enable antibody formation. If 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 antibody formation.

[0199] Using standard molecular biology techniques, recombinant expression vectors are prepared, transfected into host cells, transformants are selected, the host cells are cultured, and antibodies are recovered from the culture medium. For example, some antibodies may be isolated by affinity chromatography using a matrix coupled to protein A or protein G.

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

[0201] Pharmaceutical compositions and therapeutic methods Other aspects of this disclosure provide compositions including pharmaceutical compositions comprising any one of the humanized anti-Bb factor antibodies described herein. Generally, a pharmaceutical composition, also referred to herein as a formulation, comprises an effective amount of any one of the humanized anti-Bb factor antibodies described herein. "Effective amount" means a dosage sufficient to produce a desired outcome, such as a reduction in adverse symptoms associated with complement-mediated disease or disorder, improvement of symptoms of complement-mediated disease or disorder, or slowing of the progression of complement-mediated disease or disorder. Generally, the desired outcome is at least a reduction in symptoms of complement-mediated disease or disorder compared to a control.

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

[0203] In pharmaceutical dosage forms, humanized anti-Bb factor antibodies may be administered in the form of their pharmaceutically acceptable salts, or they may be used alone, in appropriate linkages with other pharmaceutically active compounds, or in combination. The following methods and excipients are merely illustrative and not limiting.

[0204] With regard to oral preparations, humanized anti-Bb factor antibodies can be used alone or in combination with appropriate additives to prepare tablets, powders, granules, or capsules.

[0205] Humanized anti-Bb factor antibodies can be formulated into injection preparations by dissolving, suspending, or emulsifying the antibody in an aqueous or non-aqueous solvent; and, if desired, together with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0206] Pharmaceutical compositions containing humanized anti-Bb factor antibodies are prepared by mixing humanized anti-Bb factor antibodies of desired purity with an optional physiologically acceptable carrier, other excipients, stabilizers, surfactants, buffers, and / or isotonic agents. The acceptable carrier, other excipients, and / or stabilizers are nontoxic to the recipient at the dosage and concentration employed. In some embodiments, the composition includes buffers, antioxidants, amino acids, or combinations thereof.

[0207] Pharmaceutical compositions may be in liquid form, lyophilized form, or liquid form reconstituted from a lyophilized form, and lyophilized preparations should be reconstituted with a sterile solution before administration. The standard procedure for reconstituting a lyophilized composition is to add back a certain volume of pure water (typically equivalent to the volume removed during lyophilization); however, a solution containing an antibacterial agent may be used to produce pharmaceutical compositions for parenteral administration; Chen (1992) Drug See also Dev Ind Pharm 18, pages 1311-1354.

[0208] An isotonic agent can be encapsulated in the antibody preparation to modulate its tonicity. In some embodiments, the aqueous preparation 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 for comparison, such as a physiological saline solution or serum.

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

[0210] To protect unstable active ingredients (e.g., proteins) from destabilizing conditions during the freeze-drying process, freeze-drying protectants may also be added.

[0211] In some embodiments, the formulation comprises a humanized anti-Bb factor antibody and at least one of the active ingredients identified above (e.g., surfactants, buffers, stabilizers, isotonic agents), and essentially does not contain at least one preservative.

[0212] Humanized anti-Bb factor antibodies can be used in aerosol formulations intended for administration by inhalation. Humanized anti-Bb factor antibodies can be formulated into propellants that can tolerate pressurization.

[0213] Furthermore, humanized anti-Bb factor antibodies can be prepared as suppositories by mixing them with various bases such as emulsifying agents or water-soluble bases. Humanized anti-Bb factor antibodies can then be administered rectally via suppositories.

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

[0215] Intranasal preparations will typically contain a vehicle that does not cause inflammation of the nasal mucosa and does not significantly impair ciliary function. Diluents such as water, aqueous saline, or other known substances may be used. Nasal preparations may also contain preservatives. Surfactants may be present to enhance the absorption of humanized anti-Bb factor antibodies by the nasal mucosa.

[0216] Humanized anti-Bb factor antibodies can be administered as injectable formulations. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in a liquid vehicle prior to injection are also prepared. Preparations may also be emulsified, or the antibodies may be encapsulated in liposome vehicles.

[0217] In some embodiments, the humanized anti-Bb factor antibody is formulated into a controlled-release formulation. Within the scope of this disclosure, controlled release can be understood to mean any one of several sustained-release formulations.

[0218] Humanized anti-Bb factor antibodies are administered to subjects using appropriate methods and routes for drug delivery, including in vivo and ex vivo methods, as well as 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 intestinal and parenteral administration routes.

[0220] The antibodies of this disclosure may be administered to subjects using any available conventional methods and routes suitable for conventional drug delivery, including systemic or localized routes. In general, the routes of administration envisioned by this disclosure include, but are not limited to, enteral, parenteral, or inhalation routes.

[0221] In some embodiments, humanized anti-Bb factor antibodies are administered by injection and / or delivery, for example, to a site in the cerebral arteries or directly to brain tissue. Humanized anti-Bb factor antibodies may also be administered directly to a target site, for example, by biorhythmic 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 include the order of Mammalia, including carnivores (e.g., cats), herbivores (e.g., cattle, 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). It is widely used to describe organisms that fall into this category. In some embodiments, the subject has a complement system, such as mammals, fish, or invertebrates. In some embodiments, the subject is a companion animal, farm animal, labor animal, zoo animal, or laboratory animal of a mammal, fish, or invertebrate that contains a complement system. In some embodiments, the subject is a human.

[0223] "Treatment" refers to at least improvement of symptoms associated with the pathological condition that afflicts the subject, and improvement is used in a broad sense to refer to at least a reduction in the magnitude of parameters associated with the treated pathological condition, such as symptoms, including complement-mediated disorders. Therefore, treatment also includes a situation in which the pathological condition, or at least its associated symptoms and / or secondary effects, are completely inhibited, for example, prevented from occurring, halted, or terminated, so that the subject is no longer afflicted with the pathological condition, or at least the symptoms that characterize the condition.

[0224] In some embodiments, “subject” refers to mammals, including but not limited to rodents (rats, mice), non-human primates, humans, dogs, cats, and ungulates (e.g., horses, cattle, sheep, pigs, goats). Any animal having a complement system, such as mammals, fish, and certain invertebrates, is also included in these terms. Therefore, these terms include companion animals, farm animals, labor animals, zoo animals, and laboratory animals of mammals, fish, and invertebrates that contain a complement system.

[0225] The term "biological sample" encompasses a diverse range of sample types obtained from a subject and is used in diagnostic or monitoring assays. This definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures derived therefrom, or cells 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 of 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 includes solid tissue samples, tissue culture samples, and cell samples.

[0226] This disclosure provides a device or container suitable for containing a composition comprising a humanized anti-Bb factor antibody for administration to a subject. For example, the humanized anti-Bb factor antibody may be placed in a container suitable for containing a pharmaceutical composition. The container may be, for example, a bottle (e.g., having a closing device such as a cap), a blister pack (e.g., at least one pack per blister) The container may be a vial, flexible packaging (e.g., sealed Mylar or plastic bag), ampoule (for single doses in solution), dropper, syringe, thin film, and / or tube. In some embodiments, the container, such as a sterile container, contains the pharmaceutical composition of interest. In some embodiments, the container is a bottle or 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] This disclosure provides a method for treating complement-mediated diseases or disorders. The method generally involves administering an effective amount of the humanized anti-Bb factor antibody of this disclosure to a subject in need. In some embodiments, the administration of the humanized anti-Bb factor antibody modulates complement pathway activity in the cells, tissues, or fluids of the subject to treat the complement-mediated disease or disorder.

[0228] "Effective dose" refers to the amount of anti-complement factor Bb antibody that, when administered to a mammal or other subject for the treatment of a disease, is sufficient to produce such treatment for the disease. "Therapeutic effective dose" will vary depending on the anti-complement factor Bb antibody, the disease and its severity, as well as the age, weight, etc. of the subject being treated.

[0229] In some embodiments, the effective dose of the humanized anti-Bb factor antibody of this disclosure is an amount effective in reducing or inhibiting complement pathway activity in the target cells, tissues, or body fluids.

[0230] In some embodiments, the effective dose of the humanized anti-Bb factor antibody of this disclosure is an amount effective in reducing or inhibiting MAC formation in the target cells, tissues, or bodily fluids.

[0231] In some embodiments, the effective dose of the humanized anti-Bb factor antibody of this disclosure is an amount effective in reducing or inhibiting C3b / Bb-mediated cleavage of C3 in the target cells, tissues, or body fluids.

[0232] In some embodiments, the effective amount of the humanized anti-Bb factor antibody of this disclosure is an amount effective in reducing or inhibiting C3b / Bb-mediated cleavage of C3, thereby reducing the production of C3 cleavage products.

[0233] In some embodiments, the effective dose of the humanized anti-Bb factor antibody of this disclosure is an amount effective in reducing or inhibiting complement AP-mediated lysis of cells in the subject.

[0234] In some embodiments, the effective dose of the humanized anti-Bb factor antibody of this disclosure is an amount effective in reducing or inhibiting complement AP-mediated hemolysis in the target cells, tissues, or body fluids (e.g., RBC-containing body fluids).

[0235] In some embodiments, the effective amount of the humanized anti-Bb factor antibody of this disclosure is an amount effective in reducing or inhibiting anaphylatoxin production.

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

[0237] In some embodiments, the effective amount of the humanized anti-Bb factor antibody of this disclosure is the amount of the target in the detailed This is the amount effective in reducing or inhibiting AP-mediated C3b deposition on cells or tissues.

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

[0239] In some embodiments, the effective dose of the humanized anti-Bb factor antibody of this disclosure is an amount effective in reducing or inhibiting AP-mediated C3b deposition on RBCs in the subject.

[0240] In some embodiments, the humanized anti-Bb factor antibody of the Disclosure, when administered to a subject in need of it in at least one dose, reduces the amount of circulating Bb factor in the subject. For example, the humanized anti-Bb factor antibody of the Disclosure, when administered to a subject in need of it in at least one dose, can reduce the amount of circulating Bb factor 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 Bb factor in the subject in the absence of administration of the humanized anti-Bb factor antibody, or compared to the amount of circulating Bb factor in the subject before administration of the humanized anti-Bb factor antibody.

[0241] In some embodiments, the humanized anti-Bb factor antibody of the Disclosure, when administered to a subject in need of it in at least one dose, reduces the amount of Bb factor in the plasma of the subject. For example, the humanized anti-Bb factor antibody of the Disclosure, when administered to a subject in need of it in at least one dose, can reduce the amount of Bb factor in the plasma of 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 Bb factor in the plasma of the subject in the absence of administration of the humanized anti-Bb factor antibody, or compared to the amount of Bb factor in the plasma of the subject before administration of the humanized anti-Bb factor antibody.

[0242] In some embodiments, the method of the present disclosure for treating a subject having a complement-mediated disease or disorder includes administering to the subject a pharmaceutical composition comprising: a) the humanized anti-Bb factor 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 may be by any route known to those skilled in the art, including those disclosed herein. In some embodiments, administration is intravenous. In some embodiments, administration is intrathecal. In some embodiments, administration is intramuscular. In some embodiments, administration is subcutaneous.

[0243] Complement-mediated diseases and disorders suitable for treatment with the humanized anti-Bb factor antibody of this disclosure include diseases and disorders associated with alternative complement pathways. Investigations in preclinical animal models and clinical trials have shown that alternative pathways play a crucial role in the development of tissue injury and the pathogenesis of several conditions (Holers et al., Immunological Reviews). 223:pp. 300-316; Cao et al., (2016) Haematologica 101(11):pp. 1319-1326; Schubart et al., (2019) PNAS 116(16):pp. 7926-7931; Thurman, (2015) Am J Kidney Dis 65(1):pp. 156-168; Vriese et al., (2015) Am J Kidney Dis 65(1):pp. 156-168; and Gold et al., (2006) Nat. Genet. 38(4):pp. 458-462). In some embodiments, complement-mediated diseases suitable for treatment with the humanized anti-Bb factor antibody of this disclosure are IgA kidney This includes, but is not limited to, Berger'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 gnosis, spontaneous abortion, microimmunovasculitis, epidermolysis bullosa, recurrent miscarriage, and traumatic brain injury.

[0244] The humanized anti-Bb factor antibody of this disclosure may be administered to subjects in need, either alone (e.g., as monotherapy) or in combination with at least one additional therapeutic agent.

[0245] As used herein, “in combination with” means, for example, that the first compound is administered throughout the entire course of administration of the second compound; that the first compound is administered for a period overlapping with the administration of the second compound, for example, that the administration of the first compound begins before the administration of the second compound and ends before the administration of the second compound ends; that the administration of the second compound begins before the administration of the first compound and ends before the administration of the first compound ends; that the administration of the first compound begins before the administration of the second compound begins and ends before the administration of the first compound ends; or that the administration of the second compound begins before the administration of the first compound begins and ends before the administration of the second compound ends. Therefore, “in combination with” may also mean regimens involving the administration of two or more compounds. As used herein, “in combination with” also means 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 forms.

[0246] Appropriate subjects for treatment with humanized anti-Bb factor antibodies include subjects diagnosed with complement-mediated disorders; subjects at greater risk than the general population of developing complement-mediated disorders (e.g., subjects with a genetic predisposition to developing complement-mediated disorders); subjects with any one of the complement-mediated disorders listed above. In some embodiments, subjects are adults. In some embodiments, subjects are human children. [Examples]

[0247] Humanization of mouse monoclonal anti-Bb factor antibodies Variable region genes from parental anti-Bb factor antibody (see Table 1) hybridomas were amplified, cloned, and sequenced, leading to 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 the supernatant from transfected cell cultures. Kinetic experiments were conducted 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 performed using HBS-P+ running buffer (pH 7.4) (GE Healthcare, Little Chalfont, UK). I ran in 5℃ weather.

[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, which is the theoretical value for obtaining an Rmax of approximately 50 RU. The surface was then stabilized. To minimize any potential material transport limitations, single-cycle kinetic data was obtained using Bb factor (CompTech, Tyler, USA) as the analyte at a flow rate of 30 μl / min. Surface and analyte stability over kinetic cycles was checked by performing multiple repetitions with a reference chimeric antibody. The signal from reference channel Fc1 (no antibody) was subtracted from those of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. A four-point 2x dilution range of Bb factor from 0.78 nM to 6.25 nM was used without regeneration between concentrations. The association phase was monitored for 200 seconds each for four injections of Bb factor at increasing concentrations, and the dissociation phase was measured for a single 200-second period after the final injection of Bb factor. Regeneration of the protein A surface was performed using two injections of 10 mM glycine-HCl pH 1.5.

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

[0252] [Table 7]

[0253] Biacore analysis shows that all humanized variants bind to the Bb factor, however, in all cases, relative K D This shows a difference of more than 2 times compared to chimeras, and some This suggested a loss of binding affinity. 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 of redesigned variants and single-cycle kinetic analysis Five control antibodies (VH0 / Vκ6, VH0 / Vκ7, VH5 / Vκ0, VH6 / Vκ0, VH7 / Vκ0) and combinations of humanized heavy and light chains (a total of eight humanized pairs, Table 8) were transiently transfected into HEK EBNA adherent cells (ATCC® catalog number CRL-10852®) using the PEI transfection method. IgG supernatant titers were monitored by IgG ELISA (Table 9), and the transfected cells were cultured for up to 10 days before the supernatant was harvested.

[0255] [Table 8]

[0256] Single-cycle kinetics using cell culture supernatant were performed as described above. Sensorgrams and fitted data related to 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 twice the amount of the reference chimeric antibody. K D By dividing this by the humanized variant assayed in the same experiment, the relative K D The result 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 supernatant using 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. The antibodies were then analyzed using OD based on their predicted amino acid sequence extinction coefficient. 280nm The antibodies were quantified by SDS-PAGE, which involved loading 1 μg of each antibody onto a gel (Figure 3). Bands corresponding to the profiles of typical antibodies were observed.

[0259] Multicycle kinetic analysis of antibodies To demonstrate precise affinity for the Bb factor, Biacore T200 Multicycle kinetic analysis was performed on purified chimeric antibodies and five lead antibodies using a Biacore T200 instrument (serial number 1909913) running 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, the antibodies were transferred to a Protein A chip (GE Healthcare, Little The proteins were loaded onto Fc2, Fc3, and Fc4 of Chalfont (UK). IgG was captured at a flow rate of 10 μl / min to give an immobilization level (RL) of approximately 63 RU, which is the theoretical value for obtaining an Rmax of approximately 50 RU. The surface was then stabilized. To minimize any potential mass transport effects, kinetic data were obtained using factor Bb as the analyte at a flow rate of 30 μl / min. Multiple blank repetitions and single-concentration analyte repetitions were programmed into kinetic runs to check the stability of both the surface and the analyte over kinetic cycles. For kinetic analysis, a 2-fold dilution range from 12.5 nM to 0.391 nM of factor Bb was selected. The association phase of factor Bb was monitored for 360 seconds, and the dissociation phase for 600 seconds. Regeneration of the protein A surface was performed using two injections of 10 mM glycine-HCl pH 1.5. The signal from reference channel Fc1 was subtracted from those of Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface, and the overall Rmax parameter was used in the one-to-one binding model.

[0260] Sensorgrams and fitted data regarding the binding of chimeric antibodies and humanized variants to the Bb factor are shown in Figures 4A-4C. Humanized variant K D By dividing this by the number of chimeric antibodies on the same chip, the relative K D The following was calculated. The kinetic parameters measured for the interaction of the Bb factor with the chimeric antibody and humanized variants are shown in Table 10. The two humanized variants, VH4 / Vκ6 and VH4 / Vκ7 (bold), showed a relative K of less than twice that of the reference chimeric antibody. D This was shown.

[0261] [Table 10]

[0262] Bb factor competition ELISA Purified lead variants and chimeric antibodies were tested for their binding to the Bb factor using competition against the mouse parent antibody.

[0263] Bb factor was diluted to 1.0 μg / ml in 1×PBS, and 100 μl / well was coated onto a 96-well ELISA plate overnight at 4°C. The following day, the plate was blocked at room temperature for 2 hours with 1% casein / PBS, and then washed twice with PBS pH 7.4. In the 96-well diluted plate, fixed-concentration mouse parent antibody (final concentration 0.5 μg / ml) was added in equal volumes to a 4-fold titration series of test antibody diluted in blocking buffer (starting at 45 μg / ml and decreasing to a final concentration of 0.01 μg / ml). 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 100 μl of anti-mouse IgG Fc-specific HRP (Sigma, Dorset, UK) diluted 1:1000 in PBS-T was applied at room temperature for 1 hour to detect bound mouse antibodies. For color development, the plate was washed three times with PBS-T, then 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 using a DYNEX® plate reader at 450 nm.

[0264] The results are plotted and shown in Figure 5. The IC50 value was calculated for each variant, and the relative IC50 value was 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 an IC50 value within twice that of the parent antibody.

[0265] [Table 11] [Examples]

[0266] Activity of humanized variants Inhibition of WIESLAB® AP by humanized variants of anti-Bb factor in human serum The ability of 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 invasion complex (MAC) deposition served as the readout. 5.56% normal human serum (NHS) was incubated with dilution series of the parent antibody and humanized variants, along with a human IgG4 control antibody starting at 100 μg / mL. OD405nm was measured and compared to kit-positive and negative controls. Data were plotted against plate-positive controls (Figure 6A). All humanized variants showed inhibition of AP-mediated MAC deposition in human serum, similar to the parent 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 EGTA-containing buffer that inhibits the classical pathway. Dilution series of parental antibodies and humanized variants starting at 200 μg / mL were used with 20% human serum and 10 × 10⁻¹⁰⁴ 6 The solution was incubated with rabbit erythrocytes (RBCs) at 37°C for 1 hour. The absorbance of the supernatant at 540 nm was measured, and the amount of lysis was determined by subtracting the background absorbance in a control well containing ethylenediaminetetraacetic acid (EDTA). The results are shown in Figure 6B. This is shown in Figure 6C. In Figure 6C, 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] Conjugation of parental antibodies and humanized variants to cynomolgus monkey Bb factor To ensure that the humanization process does not affect species cross-reactivity, the parent antibody and humanized derivative were tested for binding to human and cynomolgus monkey Bb factor using biolayer interferometry (BLI) with Octet Red. Briefly, the biotinylated antibody was loaded onto an SA biosensor equilibrated in PBS, 0.1% BSA, and 0.02% Tween-20 (assay buffer). After a 60-second baseline in the 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 Bb factor (in-house purified) was measured for 300 seconds, followed by 300 seconds of dissociation. Kinetic parameters were calculated using Octet Analysis software with a 1:1 binding model. The data are summarized in Table 13, demonstrating that humanization does not affect cross-reactivity to Bb factor from cynomolgus monkeys.

[0270] [Table 13]

[0271] V to human and cynomolgus monkey Bb factor H 4 / V K 6-IgG4v2 and V H 6 / V K 7-IgG4v2 binding To determine whether modification of the Fc portion of the antibody affects affinity and species cross-reactivity, V H 4 / V K 6-IgG4v2 and V H 6 / V K 7-IgG4v2 is used to identify their respective parent antibodies V H 4 / V K 6 and V H 6 / V KIn parallel with step 7, their ability to bind to human and cynomolgus monkey Bb factors was tested by BLI. VH4 / VK6-IgG4v2 and VH6 / VK7-IgG4v2 contained mutations in the Fc region that increased affinity for the Fc receptor. The experiments were carried out as described in the examples above, and the results are summarized in Table 14. As expected, the Fc modification did not affect binding.

[0272] [Table 14]

[0273] V against factor Bb (the activated form of factor B) H 4 / V K 6 and V H 6 / V K 7 Specificity V binds to zymogen factor B and factor Bb from both humans and cynomolgus monkeys. H 4 / V K 6 and V H 6 / V KThe ability of factor 7 was determined by surface plasmon resonance using a Biacore T200. Human factor B and factor Bb were purchased from Comptech, and cynomolgus monkey factor B and factor Bb were purified in-house. Briefly, monoclonal antibodies were captured on a Biacore Series S Protein A tip in HBSP+ (10 mM HEPES, 150 mM NaCl, 0.05% P20 pH 7.4) at a flow rate of 30 μL / min. Five concentration series of each analyte were tested for binding using single-cycle kinetics with a contact time of 180 seconds per concentration followed by 600 seconds of dissociation at 25°C and a flow rate of 60 μL / min. Both human and cynomolgus monkey factor B were started at a concentration of 500 nM, followed by 2-fold dilutions, while human and cynomolgus monkey factor Bb were started at concentrations of 30 nM and 150 nM, respectively, followed by 2-fold dilutions. The data were analyzed using Biacore evaluation software with a 1:1 binding model. Sensorgrams are shown in Figures 7A–7D. The results are summarized in Table 15. In short, both humanized variants showed nearly 10-fold higher affinity for human Bb factor compared to cynomolgus monkey Bb factor. Binding to human or cynomolgus monkey B factor was almost undetectable, and the small signals observed were dominated by nonspecific binding.

[0274] [Table 15]

[0275] Binding of chimeric parents and representative humanized variants to various complement proteins. To ensure that humanization does not introduce nonspecific binding or cross-reactivity to other complement or plasma proteins, chimeric parent antibodies 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 overnight at 4°C on ELISA plates at 2.5 μg / mL in PBS. The plates were then blocked with casein at room temperature for 1 hour, washed four times with 1× DPBS / 0.05% Tween-20, followed by a single wash with 1× DPBS. Serial dilutions starting at 50 μg / ml of biotinylated chimeric parent or VH6 / VK7-IgG4v2 were added to plates in PBS / 0.1% casein / 0.1% Tween-20 and incubated at room temperature for 2 hours. The plates were 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 plates and incubated at room temperature for 30 minutes. The plates were washed again, and Ultra TMP ELISA (Thermo 34028) was added to the plates for 1 minute, followed by a stop solution. OD450nm was read with a plate reader, and background at 620nm was subtracted. Both the chimeric parent (Figure 8A) and the humanized variant (Figure 8B) showed complete specificity to factor Bb. [Examples]

[0276] V produced 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, standard methods were used to determine whether it behaves similarly by transient expression in HEK cells or by stable expression in CHO cells. H 6 / V K We created 7-IgG4v2.

[0277] The complement system alternative pathway (CAP) and classical pathway (CCP) activity of the antibody was determined according to the manufacturer's instructions for use, respectively. The results were determined using the Pathway and Classical Pathway WIESLAB® kits. To determine CAP activity, antibodies were tested in 5.56% normal human serum (Figure 9A) and 5.56% normal cynomolgus monkey serum (Figure 9B). To determine CCP activity, antibodies were tested in 1% normal human serum (Figure 10A) and 1% normal cynomolgus monkey serum (Figure 10B). OD 405nm The values ​​were measured, and the results were normalized to the serum activity prior to antibody injection. V produced in CHO and HEK 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. V produced in CHO H 6 / V K 7-IgG4v2 is a IgG4 produced in HEK. H 6 / V K It showed similar efficacy to 7-IgG4v2. V produced in CHO and HEK H 6 / V K AP pathway-mediated hemolysis of 7-IgG4v2 was also compared. Hemolysis was assessed using 20% ​​normal human serum and rabbit erythrocytes 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 a control well containing ethylenediaminetetraacetic acid (EDTA). V produced in CHO and HEK H 6 / V K7-IgG4v2 showed a similar percentage of hemolysis (Figure 11).

[0278] V produced by CHO and HEK H 6 / V K Multicycle kinetic analysis was performed on the 7-IgG4v2 antibody using biolayer interferometry. 10 μg of antibody was added to PBS + 0.02% Tween20, 0.1% BSA, and 0.05% sodium azide. The solution was diluted to / mL and loaded onto an anti-hIgG Fc sensor (pre-equilibriumized in the same buffer) for 90 seconds, followed by a 60-second baseline in the buffer. Binding of a concentration series of human Bb factor (Complement Technologies, #A155) ranging from 100 nM to 0.14 nM at 2-fold dilutions was measured in the same buffer during a 300-second association, followed by a 300-second dissociation step. Binding curves and fitted data for both antibodies, as well as calculated K values, were obtained. d This is shown in Figure 12.

[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): pp. 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: pp. 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: pp. 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] In this Specification, all publications, patents, patent applications, publications, and database entries (e.g., sequence database entries) referenced in, for example, the sections of Background, Overview, Detailed Description, Examples, and / or References are incorporated by reference as a whole, as if each individual publication, patent, patent application, publication, and database entry were incorporated by reference specifically and individually in this Specification. In any conflict, the application, including any definitions herein, shall prevail.

[0281] Equivalents and range Those skilled in the art will be able to recognize or verify many equivalents of the embodiments described herein by simply using routine experiments. The scope of this disclosure is The above explanation is not intended to be the sole limiting factor, but rather as described in the attached claims.

[0282] Articles such as “a,” “an,” and “the” can mean at least one unless it is shown otherwise or the context makes otherwise obvious. Claims or descriptions containing “or” among two or more members of a group are considered to be valid when one, more than one, or all of the group members are present, unless it is shown otherwise or the context makes otherwise obvious. Disclosures of groups containing “or” among two or more group members provide 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 simplicity, such embodiments are not individually detailed herein, but it will be understood that each of these embodiments is provided herein, specifically claimed or discarded.

[0283] This disclosure should be understood to encompass all variations, combinations, and permutations of at least one limitation, element, clause, or descriptive term from at least one of the claims or from at least one relevant part of this description that are introduced into another claim. For example, a claim dependent on another claim may be modified to include at least one limitation found in any other claim dependent on the same basic claim. Furthermore, where a claim lists a composition, it should be understood to include, if any, methods of producing or using the composition, either according to any method of producing or using the composition disclosed herein or according to methods known in the art, unless otherwise indicated or unless it is obvious to a person skilled in the art that this would result in a contradiction or inconsistency.

[0284] When elements are presented as a list, for example in Markush group form, it should be understood that all conceivable subgroups of the elements are also disclosed, and that any element or subgroup of an element may be removed from the group. It should also be noted that the term “includes” is intended to be open and acknowledges the inclusion of additional elements or processes. Generally, when an embodiment, product, or method is referred to as including a particular element, configuration, or process, it should be understood that an embodiment, product, or method consisting of or essentially derived from such element, configuration, or process is also provided. For simplicity, such embodiments are not individually detailed herein, but it will be understood that each of these embodiments is provided, specifically claimed, or waived herein.

[0285] In the claims and in this specification above, all transitional phrases such as “comprising,” “including,” “bearing,” “having,” “containing,” “accompanying,” “holding,” and “composed of” should be understood to be open-ended, meaning that “including” is not limited. Only the transitional phrases “consisting of” and “essentially consisting of” are closed or semi-closed transitional phrases, respectively, as described in Section 2111.03 of the Manual of Patent Examining Procedures.

[0286] Where a domain is given, it includes the endpoint. Furthermore, unless otherwise shown or otherwise obvious from the context and / or the understanding of those skilled in the art, it should be understood that, in some embodiments, the values ​​expressed as a domain can take any specific value within the domain described, up to one-tenth of the lower limit of the domain, unless otherwise explicitly stated in the context. For simplicity, each value within the domain is not individually detailed herein, but it will be understood that each of these values ​​is provided herein, specifically claimed or waived. Unless otherwise shown or otherwise obvious from the context and / or the understanding of those skilled in the art, Unless otherwise specified, a value expressed as a domain can take any subdomain within a given domain, and it should be understood that the endpoint of a subdomain is expressed to the same precision as one-tenth of the lower limit of the domain.

[0287] If a website is provided, the URL address will be provided as non-browser executable code, with the period of each web address enclosed in parentheses. The actual web address will not contain parentheses.

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

[0289] The terms "approximately" and "effectively" preceding a number mean ±10% of the given number.

Claims

1. It specifically binds to the human complement factor Bb protein and contains the heavy chain variable region (V) of the amino acid sequence of SEQ ID NO:

19. Ｈ ) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 27 Ｌ Humanized antibodies, including ).

2. It specifically binds to the human complement factor Bb protein and contains the heavy chain variable region (V) of the amino acid sequence of SEQ ID NO:

17. Ｈ ) and the light chain variable region (V) containing the amino acid sequence of SEQ ID NO: 26 Ｌ Humanized antibodies, including ).

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

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

5. The humanized antibody according to claim 4, wherein the complement activity is selected from the group consisting of AP-mediated terminal membrane invasion complex (MAC) deposition, AP-mediated hemolysis, C3 fragment deposition on erythrocytes or other cell types, C3b / Bb-mediated cleavage of C3, and C3b / Bb / 3b-mediated cleavage of C5.

6. A humanized antibody according to any one of claims 1 to 5, which is a bispecific antibody or a polyspecific antibody.

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

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

9. A humanized antibody according to claim 8, comprising the IgG4 constant region or a variant thereof.

10. The humanized antibody according to claim 9, wherein the heavy chain constant region includes 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 containing one amino acid sequence from sequence numbers 32 to 34 and a light chain containing the amino acid sequence of sequence number 35.

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

39.

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

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

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

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

17. The device according to claim 16, which is an injection device.

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

19. A method for treating a subject having a complement-mediated disease or disorder, comprising the step of administering an effective amount of a humanized antibody according to any one of claims 1 to 12, a conjugate according to claim 13, or a pharmaceutical composition according to claim 14 or 15 to the subject to treat the complement-mediated disease.

20. The method according to claim 19, wherein the complement-mediated disease is selected from the group consisting of IgA nephropathy (Berger'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 GN, spontaneous abortion, microimmunovasculitis, epidermolysis bullosa, recurrent miscarriage, and traumatic brain injury.

21. A method for inhibiting complement pathway activity in a target, comprising the step of administering an effective amount of a humanized antibody according to any one of claims 1 to 12, a conjugate according to claim 13, or a pharmaceutical composition according to claim 14 or 15 to the target to inhibit complement activity.

22. The method according to claim 21, relating to a patient with a complement-mediated disease or disorder.

23. The method according to claim 22, wherein the complement-mediated disease is selected from the group consisting of IgA nephropathy (Berger'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 GN, spontaneous abortion, microimmunovasculitis, epidermolysis bullosa, recurrent abortion, and traumatic brain injury.

24. The method according to any one of claims 21 to 23, wherein complement activity is selected from the group consisting of AP-mediated terminal membrane invasion complex (MAC) deposition, AP-mediated hemolysis, C3 fragment deposition on erythrocytes or other cell types, C3b / Bb-mediated cleavage of C3, and C3b / Bb / 3b-mediated cleavage of C5.

25. The method according to any one of claims 19 to 24, further comprising the step of administering a therapeutic agent to a target.

26. The method according to any one of claims 19 to 25, wherein the administration is intravenous, subcutaneous, or intramuscular.

27. A nucleic acid or set of nucleic acids that encodes or collectively encodes a humanized antibody according to any one of claims 1 to 12.

28. A vector or vector set comprising the nucleic acid or nucleic acid set described in claim 27.

29. A cell expressing a humanized antibody according to any one of claims 1 to 3, a nucleic acid or nucleic acid set according to claim 27, or a vector or vector set according to claim 28.

30. The cell according to claim 29, which is a mammalian cell.

31. The cells according to claim 30, wherein the mammalian cells are selected from the group consisting of human fetal kidney (HEK) cells, Chinese hamster ovary (CHO) cells, NS0 myeloma cells, SP2 cells, COS cells, and mammary epithelial cells.

32. A method for producing a humanized antibody, comprising the step of culturing cells according to any one of claims 29 to 31 to produce a humanized antibody.

33. The method according to claim 32, further comprising the step of isolating a humanized antibody.

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