Anti-complement Bb factor antibody and its use

Abstract

Providing an anti-complement Bb factor antibody and its use. [Solution] This disclosure provides an anti-complement factor Bb antibody and a composition comprising the antibody. The anti-Bb antibody is useful for treating complement-mediated disorders. This disclosure provides a method for treating complement-mediated disorders. In some cases, the antibody is at least 10 -8 It binds to the human complement Bb protein with M affinity. In some cases, the antibody inhibits C3b / Bb-mediated cleavage of C3. In some cases, the antibody contains a humanized light chain framework region. In some cases, the antibody contains a humanized heavy chain framework region. In some cases, the antibody contains both a humanized light chain framework region and a humanized heavy chain framework region.

Description

[Technical Field]

[0001] cross reference This application claims the benefits of U.S. Provisional Patent Application No. 62 / 317,897, filed on April 4, 2016, which is incorporated herein by reference in its entirety.

[0002] Introduction The complement system is part of the innate immune system. Its primary role is to "complement" the ability of antibodies and phagocytic cells to remove harmful pathogens from an organism. However, in a wide variety of pathological contexts, from autoimmune diseases to organ transplantation, abnormal activation of the complement system occurs, leading to damage to host tissues. Several anti-complement therapies are currently in clinical development to prevent damage caused by abnormal complement system activation.

[0003] The complement system comprises three distinct upstream activation pathways, all converging on a common final pathway. Two of these pathways are induced by specific and distinct mechanisms. The classical pathway (CP) is involved when antibodies bind to antigens, and the lectin pathway (LP) is activated by carbohydrate residues on the surface of pathogens. The alternative pathway (AP), referred to as the "AP checkover," is continuously active at the basal level; its activity is unique in that it can be greatly increased by diverse signals on the foreign body surface and damaged cells via a positive feedback amplification loop. The main driving factor of the AP amplification loop is AP convertase (C3bBb). This enzyme is formed when the enzyme precursor factor B is cleaved to produce the split product fBb, which rapidly associates with surface-bound C3b to form the active enzyme C3bBb. Subsequently, C3bBb continues to cleave additional molecules of the central C3 protein, leading to the formation of opsonins (C3b, iC3b), anaphylatoxins (C3a and C5a), and the final soluble complex (MAC; C5b-9). [Overview of the Initiative] [Means for solving the problem]

[0004] overview This disclosure provides an anti-complement factor Bb antibody and a composition containing said antibody. The anti-Bb antibody is useful for treating complement-mediated disorders. This disclosure also provides a method for treating complement-mediated disorders.

[0005] This disclosure provides humanized antibodies specific to complement factor Bb, wherein the antibodies include: a) an antibody containing a light chain complementarity determining region (CDR) of the antibody light chain variable (VL) region containing amino acid sequence SEQ ID NO: 7; b) an antibody containing a light chain CDR of the antibody heavy chain variable (VH) region containing amino acid sequence SEQ ID NO: 8; c) an antibody containing a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO: 15; d) an antibody containing a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO: 16; e) an antibody containing a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO: 23; f) an antibody containing a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO: 24; g) an antibody containing a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO: 31; h) an antibody containing a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO: 32; i) amino acid sequence SEQ ID The following group is selected: (i) an antibody containing the light chain CDR of the VL region of an antibody containing NO:39; (j) an antibody containing the heavy chain CDR of the VH region of an antibody containing the amino acid sequence SEQ ID NO:40; (k) an antibody containing the light chain CDR of the VL region of an antibody containing the amino acid sequence SEQ ID NO:47; and (l) an antibody containing the heavy chain CDR of the VH region of an antibody containing the amino acid sequence SEQ ID NO:48. In the case of part, the antibody is an antibody comprising a) a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO:7 and a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO:8; b) an antibody comprising a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO:15 and a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO:16; c) an antibody comprising a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO:23 and a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO:24; d) an antibody comprising a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO:31 and a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO:32; e) an antibody comprising a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO:39 and a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO:40; and f) amino acid sequence SEQ ID The antibody is selected from the group consisting of an antibody containing a light chain CDR in the VL region containing NO:47, and an antibody containing a heavy chain CDR in the VH region containing the amino acid sequence SEQ ID NO:48. This disclosure provides a humanized antibody specific to complement factor Bb, wherein the antibody comprises: a) an antibody containing a light chain variable region containing amino acid sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; b) an antibody containing a heavy chain variable region containing amino acid sequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; c) an antibody containing amino acid sequences SEQ ID NO:9, SEQ ID NO:10, and SEQ d) Antibodies containing a light chain variable region including ID NO:11; d) Antibodies containing a heavy chain variable region including amino acid sequences SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; e) Antibodies containing a light chain variable region including amino acid sequences SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; f) Antibodies containing a heavy chain variable region including amino acid sequences SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; g) Antibodies containing amino acid sequences SEQ ID NO:25, SEQ ID The following antibodies are selected from the group: h) an antibody containing a light chain variable region including NO:26 and SEQ ID NO:27; i) an antibody containing a heavy chain variable region including amino acid sequences SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; j) an antibody containing a light chain variable region including amino acid sequences SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35; k) an antibody containing a light chain variable region including amino acid sequences SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38; and l) an antibody containing a heavy chain variable region including amino acid sequences SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46. In some cases, the antibody is a) CDR-L1 having amino acid sequence SEQ ID NO:1, CDR-L2 having amino acid sequence SEQ ID NO:2, CDR-L3 having amino acid sequence SEQ ID NO:3, amino acid sequence SEQ ID a) an antibody containing CDR-H1 having NO:4, CDR-H2 having amino acid sequence SEQ ID NO:5, and CDR-H3 having amino acid sequence SEQ ID NO:6; b) an antibody containing CDR-L1 having amino acid sequence SEQ ID NO:9, CDR-L2 having amino acid sequence SEQ ID NO:10, CDR-L3 having amino acid sequence SEQ ID NO:11, CDR-H1 having amino acid sequence SEQ ID NO:12, CDR-H2 having amino acid sequence SEQ ID NO:13, and CDR-H3 having amino acid sequence SEQ ID NO:14; c) an antibody containing CDR-L1 having amino acid sequence SEQ ID NO:17, CDR-L2 having amino acid sequence SEQ ID NO:18, CDR-L3 having amino acid sequence SEQ ID NO:19, CDR-H1 having amino acid sequence SEQ ID NO:20, CDR-H2 having amino acid sequence SEQ ID NO:21, and amino acid sequence SEQ ID Antibodies containing CDR-H3 having NO:22; d) CDR-L1 having amino acid sequence SEQ ID NO:25, CDR-L2 having amino acid sequence SEQ ID NO:26, CDR-L3 having amino acid sequence SEQ ID NO:27, CDR-H1 having amino acid sequence SEQ ID NO:28, CDR-H2 having amino acid sequence SEQ ID NO:29, and amino acid sequence S The following group is selected: (a) an antibody containing CDR-H3 having EQ ID NO:30; (e) an antibody containing CDR-L1 having amino acid sequence SEQ ID NO:33, CDR-L2 having amino acid sequence SEQ ID NO:34, CDR-L3 having amino acid sequence SEQ ID NO:35, CDR-H1 having amino acid sequence SEQ ID NO:36, CDR-H2 having amino acid sequence SEQ ID NO:37, and CDR-H3 having amino acid sequence SEQ ID NO:38; and (f) an antibody containing CDR-L1 having amino acid sequence SEQ ID NO:41, CDR-L2 having amino acid sequence SEQ ID NO:42, CDR-L3 having amino acid sequence SEQ ID NO:43, CDR-H1 having amino acid sequence SEQ ID NO:44, CDR-H2 having amino acid sequence SEQ ID NO:45, and CDR-H3 having amino acid sequence SEQ ID NO:46. In some cases, the antibody is at least 10 -8 It binds to the human complement Bb protein with M affinity. In some cases, the antibody inhibits C3b / Bb-mediated cleavage of C3. In some cases, the antibody contains a humanized light chain framework region. In some cases, the antibody contains a humanized heavy chain framework region. In some cases, the antibody contains both a humanized light chain framework region and a humanized heavy chain framework region. In some cases, the antibody is selected from the group consisting of Ig monomers, Fab fragments, F(ab')2 fragments, Fd fragments, scFv, scAb, dAb, Fv, single-domain heavy chain antibodies, and single-domain light chain antibodies. In some cases, the antibody contains light chain and heavy chain regions present in separate polypeptides. In some cases, the antibody contains light chain and heavy chain regions present in a single polypeptide.

[0006] This disclosure provides a pharmaceutical composition comprising an antibody as described in the preceding paragraph or elsewhere in this specification, and a pharmaceutically acceptable excipient. This disclosure also provides a sterile container for the pharmaceutical composition.

[0007] This disclosure provides a method for inhibiting C3b / Bb-mediated cleavage of C3 in an individual requiring inhibition, the method comprising administering to the individual an effective amount of an antibody described above or elsewhere in this specification, or an effective amount of a pharmaceutical composition described above or elsewhere in this specification. In some cases, the administration is intravenous. In some cases, the administration is subcutaneous. In some cases, the administration is intramuscular. This disclosure provides a method for treating an individual having a complement-mediated disease or disorder, the method comprising administering to the individual an effective amount of an antibody described above or elsewhere in this specification, or an effective amount of a pharmaceutical composition described above or elsewhere in this specification. In some cases, the administration is intravenous. In some cases, the administration is subcutaneous. In some cases, the administration is intramuscular. In some cases, administration results in outcomes selected from the group consisting of: a) inhibition of complement alternative pathway (AP) activity; b) inhibition of membrane invasion complex formation; c) inhibition of C3b / Bb-mediated cleavage of C3; d) reduction of C3a and / or C3b levels in body fluids or tissues; e) inhibition of factor B cleavage; f) inhibition of AP-mediated cytolysis; g) inhibition of AP-mediated hemolysis; h) inhibition of AP-mediated deposition of C3b, C3d, or other C3 cleavage products onto cells or tissues; i) inhibition of AP-mediated deposition of C3b onto red blood cells; j) reduction of circulating factor Bb in the individual; k) reduction of plasma factor Bb in the individual; and l) inhibition of anaphylatoxin production. The present invention provides, for example, the following items: (Item 1) A humanized antibody specific to complement factor Bb, wherein the antibody is a) An antibody containing a light chain complementarity determining region (CDR) in the antibody light chain variable (VL) region, including amino acid sequence SEQ ID NO:7; b) An antibody containing a light chain CDR in the variable heavy chain (VH) region of the antibody containing amino acid sequence SEQ ID NO:8; c) An antibody containing the light chain CDR of the VL region of the antibody containing amino acid sequence SEQ ID NO:15; d) An antibody containing the heavy chain CDR of the antibody VH region, which includes the amino acid sequence SEQ ID NO:16; e) An antibody containing the light chain CDR of the VL region of the antibody containing amino acid sequence SEQ ID NO:23; f) An antibody containing the heavy chain CDR of the VH region of the antibody containing amino acid sequence SEQ ID NO:24; g) An antibody containing the light chain CDR of the VL region of the antibody containing the amino acid sequence SEQ ID NO:31; h) An antibody containing the heavy chain CDR of the VH region of the antibody containing amino acid sequence SEQ ID NO:32; i) An antibody containing the light chain CDR of the antibody VL region, which includes the amino acid sequence SEQ ID NO:39; j) An antibody containing the heavy chain CDR of the VH region of the antibody with amino acid sequence SEQ ID NO:40; k) An antibody containing the light chain CDR of the VL region of the antibody containing the amino acid sequence SEQ ID NO:47; and l) The humanized antibody selected from the group consisting of an antibody containing the heavy chain CDR of the antibody VH region containing the amino acid sequence SEQ ID NO:48. (Item 2) The aforementioned antibody is a) An antibody comprising a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO:7, and a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO:8; b) An antibody containing a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO:15, and a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO:16; c) An antibody containing a light chain CDR in the VL region of the antibody containing amino acid sequence SEQ ID NO:23, and a heavy chain CDR in the VH region of the antibody containing amino acid sequence SEQ ID NO:24; d) An antibody containing a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO:31, and a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO:32; e) An antibody comprising a light chain CDR of the antibody VL region containing amino acid sequence SEQ ID NO:39, and a heavy chain CDR of the antibody VH region containing amino acid sequence SEQ ID NO:40; and f) An antibody containing a light chain CDR of the VL region of the antibody with amino acid sequence SEQ ID NO:47, and a heavy chain CDR of the VH region of the antibody with amino acid sequence SEQ ID NO:48. A humanized antibody selected from the group consisting of the following, as described in item 1. (Item 3) A humanized antibody specific to complement factor Bb, wherein the antibody is a) Amino acid sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID An antibody containing a light chain variable region including NO:3; b) Amino acid sequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID An antibody containing a heavy chain variable region including NO:6; c) Antibodies containing light chain variable regions including amino acid sequences SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; d) Antibodies containing heavy chain variable regions including amino acid sequences SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; e) Antibodies containing light chain variable regions including amino acid sequences SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; f) Antibodies containing heavy chain variable regions including amino acid sequences SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; g) An antibody containing a light chain variable region including amino acid sequences SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; h) Antibodies containing heavy chain variable regions including amino acid sequences SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; i) Antibodies containing light chain variable regions including amino acid sequences SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35; j) Antibodies containing heavy chain variable regions including amino acid sequences SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38; k) Antibodies containing light chain variable regions including amino acid sequences SEQ ID NO:41, SEQ ID NO:42, and SEQ ID NO:43; and l) An antibody containing a heavy chain variable region including amino acid sequences SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46. A humanized antibody selected from the group consisting of the following. (Item 4) The aforementioned antibody is a) Antibodies containing CDR-L1 having amino acid sequence SEQ ID NO:1, CDR-L2 having amino acid sequence SEQ ID NO:2, CDR-L3 having amino acid sequence SEQ ID NO:3, CDR-H1 having amino acid sequence SEQ ID NO:4, CDR-H2 having amino acid sequence SEQ ID NO:5, and CDR-H3 having amino acid sequence SEQ ID NO:6; b) CDR-L1 having amino acid sequence SEQ ID NO:9, CDR-L2 having amino acid sequence SEQ ID NO:10, CDR-L3 having amino acid sequence SEQ ID NO:11, CDR-H1 having amino acid sequence SEQ ID NO:12, CDR-H2 having amino acid sequence SEQ ID NO:13, and amino acid sequence SEQ ID An antibody containing CDR-H3 with NO:14; c) CDR-L1 having amino acid sequence SEQ ID NO:17, amino acid sequence SEQ Antibodies containing CDR-L2 with ID NO:18, CDR-L3 with amino acid sequence SEQ ID NO:19, CDR-H1 with amino acid sequence SEQ ID NO:20, CDR-H2 with amino acid sequence SEQ ID NO:21, and CDR-H3 with amino acid sequence SEQ ID NO:22; d) CDR-L1 having amino acid sequence SEQ ID NO:25, amino acid sequence SEQ Antibodies containing CDR-L2 with ID NO:26, CDR-L3 with amino acid sequence SEQ ID NO:27, CDR-H1 with amino acid sequence SEQ ID NO:28, CDR-H2 with amino acid sequence SEQ ID NO:29, and CDR-H3 with amino acid sequence SEQ ID NO:30; e) CDR-L1 having amino acid sequence SEQ ID NO:33, amino acid sequence SEQ Antibodies containing CDR-L2 having ID NO:34, CDR-L3 having amino acid sequence SEQ ID NO:35, CDR-H1 having amino acid sequence SEQ ID NO:36, CDR-H2 having amino acid sequence SEQ ID NO:37, and CDR-H3 having amino acid sequence SEQ ID NO:38; and f) CDR-L1 having amino acid sequence SEQ ID NO:41, amino acid sequence SEQ CDR-L2 with ID NO:42, CDR-L3 with amino acid sequence SEQ ID NO:43, CDR-H1 with amino acid sequence SEQ ID NO:44, CDR-H2 with amino acid sequence SEQ ID NO:45, and amino acid sequence SEQ ID Antibody containing CDR-H3 with NO:46 A humanized antibody as described in item 3, selected from the group consisting of the following. (Item 5) The aforementioned antibody contains at least 10 -8 A humanized antibody described in any one of items 1-4, which binds to the human complement Bb protein with M affinity. (Item 6) The aforementioned antibody is a humanized antibody described in any one of items 1 to 4, which inhibits C3b / Bb-mediated cleavage of C3. (Item 7) The aforementioned antibody is a humanized antibody according to any one of items 1 to 4, comprising a humanized light chain framework region. (Item 8) The aforementioned antibody is a humanized antibody as described in any one of items 1 to 4, which includes a humanized heavy chain framework region. (Item 9) The aforementioned antibody is a humanized antibody according to any one of items 1 to 4, comprising a humanized light chain framework region and a humanized heavy chain framework region. (Item 10) The antibody is a humanized antibody as described in any one of items 1 to 9, selected from the group consisting of Ig monomers, Fab fragments, F(ab')2 fragments, Fd fragments, scFv, scAb, dAb, Fv, single-domain heavy chain antibodies, and single-domain light chain antibodies. (Item 11) The antibody is a humanized antibody according to any one of items 1 to 9, comprising a light chain region and a heavy chain region present in a separate polypeptide. (Item 12) The antibody is a humanized antibody according to any one of items 1 to 9, comprising a light chain region and a heavy chain region present in a single polypeptide. (Item 13) A pharmaceutical composition comprising an antibody as described in any one of items 1 to 12, and a pharmaceutically acceptable excipient. (Item 14) A sterile container containing the pharmaceutical composition described in item 13. (Item 15) A method for inhibiting C3b / Bb-mediated cleavage of C3 in an individual requiring inhibition, the method comprising administering to the individual an effective amount of an antibody described in any one of items 1 to 12, or an effective amount of a pharmaceutical composition described in item 13. (Item 16) The administration is intravenous, as described in item 15. (Item 17) The administration is subcutaneous, as described in item 15. (Item 18) The aforementioned administration is intramuscular, as described in item 15. (Item 19) A method for treating an individual having a complement-mediated disease or disorder, the method comprising administering to the individual an effective amount of an antibody described in any one of items 1 to 12. (Item 20) The administration is intravenous, as described in item 19. (Item 21) The administration is subcutaneous, as described in item 19. (Item 22) The aforementioned administration is intramuscular, as described in item 19. (Item 23) The aforementioned administration a) Inhibition of complement alternative pathway (AP) activity; b) Inhibition of membrane invasion complex formation; c) Inhibition of C3b / Bb-mediated cleavage of C3; d) Decreased levels of C3a and / or C3b in body fluids or tissues; e) Inhibition of factor B cleavage; f) Inhibition of AP-mediated cytolysis; g) Inhibition of AP-mediated hemolysis; h) Inhibition of AP-mediated deposition of C3b, C3d, or other C3 fragmentation products onto cells or tissues; i) Inhibition of AP-mediated deposition of C3b on red blood cells; j) A decrease in the amount of circulating Bb factor in the said individual; k) A decrease in the amount of Bb factor in the plasma of the said individual; and l) Inhibition of anaphylatoxin production A method according to any one of items 15-22, which produces an outcome selected from the group consisting of the following. (Item 24) A method for inhibiting the binding of factor H to C3b / Bb in an individual requiring inhibition, the method comprising administering to the individual an effective amount of an antibody described in any one of items 1 to 12, or an effective amount of a pharmaceutical composition described in item 13. (Item 25) The administration is intravenous, as described in item 24. (Item 26) The administration is subcutaneous, as described in item 24. (Item 27) The aforementioned administration is intramuscular, as described in item 24. (Item 28) A method for reducing the amount of complement C3 in body fluids or tissues in an individual requiring reduction, the method comprising administering to the individual an effective amount of an antibody described in any one of items 1 to 12, or an effective amount of a pharmaceutical composition described in item 13. (Item 29) The administration is intravenous, as described in item 28. (Item 30) The administration is subcutaneous, as described in item 28. (Item 31) The aforementioned administration is intramuscular, as described in item 28. [Brief explanation of the drawing]

[0008] [Figure 1] This image depicts the binding of anti-Bb factor monoclonal antibodies (mAbs) to human Bb factor using an enzyme-linked immunosorbent assay (ELISA). [Figure 2] This describes the EC50 for the binding of anti-Bb factor mAbs to Bb factor. [Figure 3] This depicts the ratio of binding of anti-Bb factor mAbs to soluble B factor versus soluble B factor. [Figure 4] The ratio of binding of anti-Bb factor mAbs to soluble B factor versus soluble B factor, and the binding of anti-Bb factor mAbs to B factor by size exclusion chromatography are depicted. [Figure 5] This describes the binding affinity of anti-Bb factor mAbs to B factor and Bb factor in cynomolgus monkeys. [Figure 6] This describes the inhibition of complement alternative pathway (AP) activity by anti-Bb factor mAbs (as determined using the Wieslab ELISA kit). [Figure 7] This describes the inhibition of AP-mediated hemolysis in rabbit erythrocytes by anti-Bb factor mAbs. [Figure 8] This describes the inhibition of AP-mediated C3b deposition on rabbit erythrocytes (RBCs) by anti-Bb factor mAbs. [Figure 9] This describes the inhibition of complement AP activity by anti-Bb factor mAbs. [Figure 10] This describes the inhibition of complement AP activity in cynomolgus monkeys and humans by anti-Bb factor mAbs (Wieslab ELISA kit). [Figure 11] This describes the inhibition of AP-mediated hemolysis of rabbit erythrocytes in cynomolgus monkeys by anti-Bb factor mAbs. [Figure 12A] Table 1 provides the amino acid sequences of the VH and VL regions, as well as the complementarity-determining region (CDR), of examples of anti-Bb mAbs of this disclosure. [Figure 12B] Table 1 provides the amino acid sequences of the VH and VL regions, as well as the CDR, of examples of anti-Bb mAbs of this disclosure. [Figure 12C] Table 1 provides the amino acid sequences of the VH and VL regions, as well as the CDR, of examples of anti-Bb mAbs of this disclosure. [Figure 12D] Table 1 provides the amino acid sequences of the VH and VL regions, as well as the CDR, of examples of anti-Bb mAbs of this disclosure. [Figure 12E] Table 1 provides the amino acid sequences of the VH and VL regions, as well as the CDR, of examples of anti-Bb mAbs of this disclosure. [Figure 12F] Table 1 provides the amino acid sequences of the VH and VL regions, as well as the CDR, of examples of anti-Bb mAbs of this disclosure. [Figure 13] We are providing the amino acid sequence (SEQ ID NO: 49) of the human Bb factor. [Figure 14] This disclosure describes the inhibition of AP pathway-mediated hemolysis by the Bb factor antibody against human RBCs pretreated with CD55 / CD59 antibody. [Figure 15] This describes the inhibition of the complement alternative pathway (AP) by chimeric M10 and M4 antibodies of the Bb factor chimeric antibody. [Figure 16] This study describes the inhibition of AP pathway-mediated hemolysis by chimeric M10, an anti-Bb factor chimeric antibody, against RBCs derived from patients with paroxysmal nocturnal hemoglobinuria (PNH). [Figure 17]This paper describes the pharmacokinetics (PK) of chimeric M10 and chimeric M4 anti-Bb factor chimeric antibodies in cynomolgus monkeys. [Figure 18] This paper describes the pharmacodynamics (PD) of the anti-Bb factor chimeric antibody M10 in cynomolgus monkeys. [Figure 19] This paper describes the pharmacodynamics of the anti-Bb factor chimeric antibody M4 in cynomolgus monkeys. [Figure 20] This describes chimeric anti-Bb factor chimeric M4-induced C3 degradation in human serum in vitro. [Figure 21] This image depicts chimeric anti-Bb factor chimeric M10-induced (upper panel) and chimeric anti-Bb factor chimeric M4-induced (lower panel) C3 degradation in cynomolgus monkeys in vivo. [Figure 22] This describes the blockade of the H factor by the anti-Bb factor antibody M4, which affects the DAF (degradation acceleration factor) activity. [Figure 23] This describes the effect of anti-Bb factor M4 on the DAF activity of CD55. [Modes for carrying out the invention]

[0009] definition The terms "antibody" and "immunoglobulin" include, but are not limited to, antibodies, immunoglobulins of any isotype, or antibody fragments that retain specific binding to an antigen, including Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies (scAb), single-domain antibodies (dAb), single-domain heavy-chain antibodies, single-domain light-chain antibodies, bispecific antibodies, polyspecific antibodies, and fusion proteins containing the antigen-binding (also referred to herein as antigen-binding) portion of an antibody and a non-antibody protein. Antibodies can be detected by labeling with, for example, radioisotopes, enzymes that produce detectable products, fluorescent proteins, etc. Antibodies can be further conjugated with other parts, such as members of specific binding pairs, for example, biotin (a member of the biotin-avidin specific binding pair). Antibodies can also be conjugated to solid supports, including but not limited to polystyrene plates or beads. The term also includes other antibody fragments that retain specific binding to antigens, such as Fab', Fv, F(ab')2, and / or monoclonal antibodies. As used herein, a monoclonal antibody is an antibody produced by a group of identical cells, all of which are produced from a single cell by repeated cell replication. That is, a clone of a cell produces only one antibody species. Monoclonal antibodies can be 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). Antibodies can be monovalent or bivalent. Antibodies can be Ig monomers, which are "Y-shaped" molecules consisting of four polypeptide chains: two heavy chains and two light chains linked by disulfide bonds.

[0010] As used herein, the term “humanized immunoglobulin” refers to an immunoglobulin containing immunoglobulin portions of different origins, at least one of which contains an amino acid sequence of human origin. For example, a humanized antibody may contain a portion derived from a non-human immunoglobulin of essential specificity, such as mouse, which is chemically joined together by conventional techniques (e.g., synthetic) or prepared as a continuous polypeptide using genetic engineering techniques, and a portion derived from a human immunoglobulin sequence (e.g., chimeric immunoglobulin) (e.g., the DNA encoding the protein portion of a chimeric antibody may be expressed to produce a continuous polypeptide chain). Another example of a humanized immunoglobulin is an immunoglobulin containing one or more immunoglobulin chains containing a CDR derived from a non-human antibody and a human light chain and / or framework region derived from the light chain (e.g., a CDR graft antibody 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., WO86 / 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, 242:423-426 (1988).

[0011] For example, humanized immunoglobulins can be produced using synthetic and / or recombinant nucleic acids to prepare genes (e.g., cDNA) encoding the desired humanized strand. For example, nucleic acid (e.g., DNA) sequences encoding humanized variable regions can be constructed using PCR mutagenesis to modify DNA sequences encoding human or humanized strands, such as DNA templates derived from previous humanized variable regions (see, for example, Kamman, M., et al., Nucleic Acids Res., 17:5404 (1989); Sato, K., et al., Cancer Research, 53:851-856 (1993); Daugherty, B. Let al., Nucleic Acids Res., 19(9):2471-2476 (1991); and Lewis, A.P. and J.Sc. Rowe, Gene, 101:297-302 (1991)). Variants can also be readily produced using these or other suitable methods. For example, a cloned variable region can be mutated, and a sequence encoding a variant with desired specificity can be selected (e.g., from a phage library; e.g., Krebber et al., U.S. 5,514,5 See issue 48; Hoogenboom et al., WO93 / 06213, published April 1, 1993.

[0012] An "antibody fragment" is a portion of an intact antibody, such as the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)); domain antibodies (dAb; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and polyspecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site and a remainder "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 with antigens.

[0013] "Fv" is the minimal antibody fragment containing complete antigen recognition and antigen-binding sites. This region consists of a dimer of one heavy chain variable domain and one light chain variable domain, bound by a rigid, non-covalent bond. It is formed when the three CDRs of each variable domain interact to form V H -V L This configuration defines the antigen-binding site on the surface of the dimer. Together, 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.

[0014] The "Fab" fragment also contains the constant domain of the light chain and the 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, which contains one or more cysteines derived from the antibody hinge region. Fab'-SH is the herein designation for Fab' fragments in which the cysteine ​​residue(s) of the constant domain have a free thiol group. The F(ab')2 antibody fragment is originally produced as a pair of Fab' fragments, with a hinge cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0015] The "light chains" of antibodies (immunoglobulins) derived from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domains of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. Subclasses can be further subdivided into types, e.g., IgG2a and IgG2b.

[0016] "Single-stranded Fv", "sFv", or "scFv" antibody fragments are the V of the antibody. H and V L It contains domains, and these domains are present in a single polypeptide chain. In some cases, the Fv polypeptide is V H and V LIt further comprises a polypeptide linker between the domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenberg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0017] The term "diabody" refers to a small antibody fragment having two antigen-binding sites and the fragment comprises a heavy chain variable domain (V L ) connected to a light chain variable domain (V H ) within the same polypeptide chain (V H -V L ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains on another chain, creating two antigen-binding sites. Diabodies are well described, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.

[0018] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two agents (e.g., an antibody and an antigen), the dissociation constant (K DThe affinity can be at least 1x greater than the affinity of the antibody to an unrelated amino acid sequence, at least 2x greater, at least 3x greater, at least 4x greater, at least 5x greater, at least 6x greater, at least 7x greater, at least 8x greater, at least 9x greater, at least 10x greater, at least 20x greater, at least 30x greater, at least 40x greater, at least 50x greater, at least 60x greater, at least 70x greater, at least 80x greater, at least 90x greater, at least 100x greater, or at least 1,000x greater, or greater. The affinity of the antibody to the target protein can be, for example, about 100 nanomolars (nM) to about 0.1 nM, about 100 nM to about 1 picomolar (pM), or about 100 nM to about 1 femtomolear (fM), or greater. As used herein, the term "avidity" refers to the resistance of a complex of two or more active ingredients to dissociation after dilution. The terms "immunoreactive" and "preferentially binding" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0019] The term "binding" refers to a direct link between two molecules, including interactions such as covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding, including interactions such as salt bridges and water bridges. The anti-Bb antibodies of this disclosure specifically bind to epitopes within the complement Bb protein. "Specific binding" means at least about 10 -7 M or larger, for example, 5 x 10 -7 M, 10 -8 M, 5×10 -8 This refers to binding with affinity M and above. "Non-specific binding" refers to binding with affinity of approximately 10 -7 Joining at affinity levels less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 This refers to bonding using affinity such as M.

[0020] As used herein, the terms “CDR” or “complementarity-determining region” are intended to mean discontinuous antigen linkage sites found within the variable regions of both heavy and light chain polypeptides. CDR is described by Lefranc et al. (2003) Developmental and Comparative Immunology 27:55 (also referred to herein as "Lefranc 2003"); 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) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and is defined as including duplication or subsets of amino acid residues when compared to one another. Nevertheless, the application of any definition referring to an antibody, graft antibody, or CDR of a variant thereof is intended to be within the scope of the terms defined and used herein. The amino acid residues containing CDRs, as defined, are shown in Table 2 below for comparison. The CDRs listed in Table 1 (provided in Figures 12A-12F) are defined according to Lefranc 2003. [Table 2]

[0021] 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.

[0022] As used herein, the term “framework,” when used in relation to the antibody variable region, is intended to mean all amino acid residues outside the CDR region within the antibody variable region. While the variable region framework is generally a discontinuous amino acid sequence of approximately 100–120 amino acids in length, only those amino acids outside the CDR are intended to be referred to. As used herein, the term “framework region” is intended to mean each domain of the framework separated by the CDR.

[0023] An “isolated” antibody is one that has been identified and separated and / or recovered from its constituent elements in its natural environment. Contaminating elements in its natural environment are materials that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some cases, the antibody is purified to (1) a percentage of the antibody determined by the Lowry method that is greater than 90%, greater than 95%, or greater than 98%, for example, greater than 99%, or to a percentage that is sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer, or (3) to homogeneity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions using Coomassie blue or silver staining. Isolated antibodies include antibodies in situ within recombinant cells, because at least one component of the antibody’s natural environment would not be present. In some cases, the isolated antibody is prepared by at least one purification step.

[0024] In this specification, "polypeptide," "peptide," and "tan" are interchangeable terms. The term "protein" refers to a polymeric form of amino acids of any length, and may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone. The term includes, but is not limited to, fusion proteins having heterologous amino acid sequences, fusions having heterologous and homologous leader sequences with or without an N-terminal methionine residue, and immunologically tagged proteins.

[0025] As used herein, terms such as “treatment,” “to treat,” and “to cure” mean obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it is a partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, “treatment” encompasses any treatment of a disease in mammals, particularly in humans, and includes (a) preventing the onset of a disease in an object that is susceptible to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., stopping its development; and (c) reducing the disease, i.e., causing its regression.

[0026] The terms “individual,” “subject,” “host,” and “patient,” as used interchangeably herein, refer 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). These terms also encompass any animal with a complement system, such as mammals, fish, and certain invertebrates. Such animals include companion animals, farm animals, labor animals, zoo animals, and laboratory animals of mammals, fish, and invertebrates that contain a complement system.

[0027] The term "therapeutably effective dose" or "effective dose" refers to the amount of anti-complement Bb antibody that, when administered to a mammal or other subject for the treatment of a disease, is sufficient to produce such treatment for that disease. The "therapeutably effective dose" varies depending on the anti-complement Bb antibody, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0028] The term "biological sample" encompasses a diverse range of sample types obtained from an individual and may be used in diagnostic or monitoring assays. This definition includes blood and other fluid samples of biological origin, solid tissue samples such as biopsy specimens or tissue cultures, or cells and their offspring derived therefrom. The definition also includes samples that have been handled in any manner after their acquisition, such as by reagent treatment, solubilization, or enrichment of certain components, such as polynucleotides. The term "biological sample" encompasses clinical samples, including 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, plasma, and blood fractions such as serum. The term "biological sample" also includes solid tissue samples, tissue culture samples, and cell samples.

[0029] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described and may naturally vary as such. Since the scope of the present invention is limited only by the appended claims, it should also be understood that the terminology used herein is for the purpose of describing only specific embodiments and is not intended to be restrictive.

[0030] If a range of values ​​is provided, each intermediary value between the upper and lower limits of that range, up to one-tenth of the lower limit unit unless otherwise explicitly stated in the context, and the description thereof Any other described or intervening values ​​within a range are understood to be included within the present invention. The upper and lower limits of these smaller ranges may be independently included within those smaller ranges and, depending on any limits specifically excluded in the described range, are also included within the present invention. If a described range includes one or both of the limits, a range that excludes one or both of those included limits is also included within the present invention.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of the present invention, but preferred methods and materials are described herein. All publications referenced herein for the purpose of disclosing and describing methods and / or materials cited in connection therewith are incorporated herein by reference.

[0032] When used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple subjects unless the context clearly indicates otherwise. For example, "anti-Bb antibody (an References to “anti-Bb antibody)” include multiple such antibodies, and references to “the composition” include one or more compositions, and their equivalents known to those skilled in the art. It should be further noted that the claims may be drafted to exclude any optional elements. As such, this statement is intended to act as an antecedent against the use of exclusive or “negative” limitation terms such as “only,” “only,” etc., relating to the enumeration of elements of the claims.

[0033] Certain features of the Invention described in the context of separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, various features of the Invention described in the context of a single embodiment for brevity may also be provided individually or in any suitable partial combination. All combinations of embodiments relating to the Invention are specifically encompassed by the Invention and are disclosed herein as if every possible combination were individually and expressly disclosed. In addition, all partial combinations of various embodiments and their elements are also specifically encompassed by the Invention and are disclosed herein as if every possible combination were individually and expressly disclosed herein.

[0034] The publications discussed herein are provided only for disclosures prior to the filing date of this application. Nothing herein should be construed as an acknowledgment that the present invention is not granted prior rights to such publications on the grounds of prior art. Furthermore, the dates of the publications provided may differ from the actual publication dates and may need to be independently verified.

[0035] Detailed explanation This disclosure provides an anti-complement factor Bb antibody and a composition containing said antibody. The anti-Bb antibody is useful for treating complement-mediated disorders. This disclosure also provides a method for treating complement-mediated disorders.

[0036] Anti-complement Bb antibody This disclosure provides anti-complement Bb antibodies and pharmaceutical compositions comprising such antibodies. The anti-complement Bb antibodies of this disclosure are also referred to herein as “anti-Bb” antibodies or “anti-Bb factor” antibodies. In some cases, the anti-complement Bb antibodies of this disclosure are humanized. In some cases, the anti-complement Bb antibody of the present disclosure comprises at least one humanized light chain variable region (VL) framework region. In some cases, the anti-complement Bb antibody of the present disclosure comprises at least one humanized heavy chain variable region (VH) framework region. In some cases, the anti-complement Bb antibody of the present disclosure comprises at least one humanized VL framework region and at least one humanized VH framework region.

[0037] Humanization of a framework region(s) reduces the risk that the antibody will elicit a human anti-mouse antibody (HAMA) response in humans. HAMA responses in specific patients or during clinical trials can be monitored by methods accepted in the art to determine the immune response. Patients administered with humanized antibodies may be given immunogenicity assessments at the start of therapy administration and throughout administration. HAMA responses are measured by detecting antibodies against humanized therapeutic reagents in patient-derived serum samples using methods known to those skilled in the art, including, for example, surface plasmon resonance (BIACORE) and / or solid-phase enzyme-linked immunosorbent assay (ELISA) analysis. In many cases, the humanized anti-Bb antibodies of this disclosure do not substantially elicit a HAMA response in human subjects.

[0038] Certain amino acids derived from human variable region framework residues are selected for substitution based on their possible effects on the CDR conformation and / or binding antigen. Unnatural parallelism between the mouse CDR region and the human variable framework region can result in unnatural conformational limitations, which, unless corrected by substitution of certain amino acid residues, lead to a loss of binding affinity.

[0039] The selection of amino acid residues for substitution can, in part, be determined by computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are known in the art. Generally, molecular models are constructed from elucidated structures of immunoglobulin chains or their domains. The chain to be modeled is compared with the chain or domain of the elucidated three-dimensional structure for amino acid sequence similarity, and the chain or domain showing the greatest sequence similarity is selected as the starting point for constructing the molecular model. Chains or domains sharing at least 50% sequence identity are selected for modeling, and those sharing, for example, at least 60%, at least 70%, at least 80%, at least 90%, or higher, sequence identity are selected for modeling. The elucidated starting structure is modified to allow for differences between the actual amino acids in the immunoglobulin chain or domain being modeled and those in the starting structure. The modified structure is then assembled into a hybrid immunoglobulin. Finally, the model is refined by minimizing energy and by verifying that all atoms are at a suitable distance from each other and that the bond lengths and angles are within chemically acceptable limits.

[0040] The CDR and framework regions are defined as follows: Lefranc et al. (2003) Developmental and Comparative Immunology 27:55 (hereinafter also referred to as "Lefranc 2003"); Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md., 1987 and 1991). Alternative structural definitions have been proposed by Chothia et al., J.Mol.Biol.196:901 (1987); Nature 342:878 (1989); and J.Mol.Biol.186:651 (1989) (collectively referred to as "Chothia"). When the framework residues defined by Kabat form the structural loop residues defined by Chothia, amino acids present in mouse antibodies can be selected for substitution in humanized antibodies. Residues "proximally adjacent to the CDR region" are human This includes amino acid residues in immediate proximity to one or more CDRs in the primary sequence of the immunoglobulin chain, for example, in immediate proximity to CDRs as defined by Kabat or Chothia (see, e.g., Chothia and Lesk JMB 196:901 (1987)). These amino acids, in particular, interact with amino acids in the CDRs and, if selected from the acceptor, may deform the donor CDR and reduce its affinity. Furthermore, adjacent amino acids may directly interact with the antigen (Amit et al., Science, 233:747 (1986)), and selecting these amino acids from the donor may be desirable to preserve all antigen contact that provides affinity in the original antibody.

[0041] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M10 antibody, the CDRs as defined by Lefranc 2003. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M10 antibody, the CDRs as defined by Lefranc 2003. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M10 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M10 antibody, the CDRs as defined by Lefranc 2003. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes the framework domain (FR).

[0042] In some cases, the anti-Bb antibodies of this disclosure include a light chain variable region containing one, two, or three VL CDRs of the M10 antibody, such CDRs as defined by Kabat 1991. In some cases, the anti-Bb antibodies of this disclosure include a heavy chain variable region containing one, two, or three VH CDRs of the M10 antibody, such CDRs as defined by Kabat As defined in 1991. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M10 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M10 antibody, the CDRs as defined in Kabat 1991. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0043] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M10 antibody, the CDRs as defined by Chothia 1987. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M10 antibody, the CDRs as defined by Chothia 1987. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M10 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M10 antibody, the CDRs as defined by Chothia 1987. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0044] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody, the CDRs as defined by Lefranc 2003. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs as defined by Lefranc 2003. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs This is as defined by Lefranc 2003. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0045] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody, the CDRs as defined by Kabat 1991. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs as defined by Kabat 1991. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs as defined by Kabat 1991. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0046] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody, the CDRs as defined by Chothia 1987. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs as defined by Chothia 1987. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs as defined by Chothia 1987. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0047] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody, the CDRs as defined by Lefranc 2003. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs as defined by Lefranc 2003. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs as defined by Lefranc 2003. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0048] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody, the CDRs as defined by Kabat 1991. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs as defined by Kabat 1991. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs as defined by Kabat 1991. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0049] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody, the CDRs as defined by Chothia 1987. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs as defined by Chothia 1987. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M4 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M4 antibody, the CDRs This is as defined by Chothia 1987. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0050] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M20 antibody, the CDRs being as defined by Lefranc 2003. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M20 antibody, the CDRs being as defined by Lefranc 2003. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M20 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M20 antibody, the CDRs being as defined by Lefranc 2003. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0051] In some cases, the anti-Bb antibodies of this disclosure include a light chain variable region containing one, two, or three VL CDRs of the M20 antibody, such CDRs as defined by Kabat 1991. In some cases, the anti-Bb antibodies of this disclosure include a heavy chain variable region containing one, two, or three VH CDRs of the M20 antibody, such CDRs as defined by Kabat As defined in 1991. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M20 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M20 antibody, the CDRs as defined in Kabat 1991. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0052] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M20 antibody, the CDRs as defined by Chothia 1987. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M20 antibody, the CDRs as defined by Chothia 1987. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M20 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M20 antibody, the CDRs as defined by Chothia 1987. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0053] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M17 antibody, the CDRs as defined by Lefranc 2003. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M17 antibody, the CDRs as defined by Lefranc 2003. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M17 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M17 antibody, the CDRs as defined by Lefranc 2003. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0054] In some cases, the anti-Bb antibodies of this disclosure include a light chain variable region containing one, two, or three VL CDRs of the M17 antibody, such CDRs as defined by Kabat 1991. In some cases, the anti-Bb antibodies of this disclosure include a heavy chain variable region containing one, two, or three VH CDRs of the M17 antibody, such CDRs as defined by Kabat As defined in 1991. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M17 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M17 antibody, the CDRs as defined in Kabat 1991. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0055] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M17 antibody, the CDRs as defined by Chothia 1987. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain variable region containing one, two, or three VH CDRs of the M17 antibody, the CDRs as defined by Chothia 1987. In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region containing one, two, or three VL CDRs of the M17 antibody; and a heavy chain variable region containing one, two, or three VH CDRs of the M17 antibody, the CDRs as defined by Chothia 1987. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0056] In some cases, the anti-Bb antibody of this disclosure is SEQ ID NO:1, SEQ ID The anti-Bb antibody of this disclosure comprises a light chain region containing one, two, or three CDRs selected from NO:2 and SEQ ID NO:3. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain region containing one, two, or three CDRs selected from SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some cases, the anti-Bb antibody of this disclosure comprises a) a light chain region containing one, two, or three CDRs selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and b) a heavy chain region containing one, two, or three CDRs selected from SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0057] In some cases, the anti-Bb antibody of this disclosure is SEQ ID NO:9, SEQ ID The light chain region includes one, two, or three CDRs selected from SEQ ID NO:10 and SEQ ID NO:11. In some cases, the anti-Bb antibody of this disclosure includes a heavy chain region including one, two, or three CDRs selected from SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some cases, the anti-Bb antibody of this disclosure includes a) a light chain region including one, two, or three CDRs selected from SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; and b) SEQ ID NO:12, SEQ ID NO:13, and SEQ ID The heavy chain region comprises one, two, or three CDRs selected from NO:14. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0058] In some cases, the anti-Bb antibody of this disclosure comprises a light chain region containing one, two, or three CDRs selected from SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain region containing one, two, or three CDRs selected from SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22. In some cases, the anti-Bb antibody of this disclosure comprises a) a light chain region containing one, two, or three CDRs selected from SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; and b) a heavy chain region containing one, two, or three CDRs selected from SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Fray Includes the Mwork area.

[0059] In some cases, the anti-Bb antibody of this disclosure comprises a light chain region containing one, two, or three CDRs selected from SEQ ID NO:25, SEQ ID NO:2, and SEQ ID NO:26. In some cases, the anti-Bb antibody of this disclosure comprises a heavy chain region containing one, two, or three CDRs selected from SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29. In some cases, the anti-Bb antibody of this disclosure comprises a) a light chain region containing one, two, or three CDRs selected from SEQ ID NO:25, SEQ ID NO:2, and SEQ ID NO:26; and b) SEQ ID NO:27, SEQ ID NO:28, and SEQ ID The heavy chain region comprises one, two, or three CDRs selected from NO:29. In some of these embodiments, the anti-Bb antibody is humanized V H and / or V L Includes framework domains.

[0060] In some cases, humanized V H Framework or V L The framework is the Consensus Human Framework. The Consensus Humanization Framework is based on human immunoglobulin V L or V H This can represent the most abundant amino acid residue in the selection of a framework sequence.

[0061] V as described herein H Consensus Human V suitable for use with CDR H Non-restrictive examples of framework domains include the following (Subgroup III consensus):

[0062] a)V H FR1:EVQLVESGGGLVQPGGSLRLSCAAS(SEQ ID NO:50);

[0063] b)V H FR2:WVRQAPGKGLEWV(SEQ ID NO:51);

[0064] c)V H FR3:RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO:52); and

[0065] d)V H FR4:WGQGTLVTVSS(SEQ ID NO:54).

[0066] In some cases, V H FR3 contains amino acid substitutions at positions 71, 73, and / or 78; for example, [ka] In this context, the underlined and bolded R represents amino acid 71 (Kabat numbering); [ka] In the above, the underlined and bolded N is amino acid 73 (Kabat numbering); and [ka] In the above, the underlined and bolded L is amino acid 78 (Kabat numbering). For example, in some cases amino acid 71 is A; and / or amino acid 73 is T; and / or amino acid 78 is A. As an example, in some cases appropriate consensus humanization V H FR3 has the following amino acid sequence: [ka] Includes.

[0067] V as described herein H Consensus Human V suitable for use with CDR H Non-restrictive examples of framework domains include the following (subgroup I consensus):

[0068] a)V H FR1:QVQLVQSGAEVKKPGASVKVSCKAS(SEQ ID NO:55);

[0069] b)V H FR2:WVRQAPGQGLEWM(SEQ ID NO:56);

[0070] c)V H FR3:RVTITADTSTSTAYMELSSLRSEDTAVYYC(SEQ ID NO:57); and

[0071] d)V H FR4:WGQGTLVTVSS(SEQ ID NO:58).

[0072] V as described herein H Consensus Human V suitable for use with CDR H Non-restrictive examples of framework domains include the following (Subgroup II consensus):

[0073] a)V H FR1:QVQLQESGPGLVKPSQTLSLTCTVS(SEQ ID NO:59);

[0074] b)V H FR2:WIRQPPGKGLEWI(SEQ ID NO:60);

[0075] c)V H FR3:RVTISVDTSKNQFSLKLSSVTAADTAVYYC(SEQ ID NO:61); and

[0076] d)V H FR4:WGQGTLVTVSS(SEQ ID NO:62).

[0077] V as described herein L Consensus Human V suitable for use with CDR LNon-limiting examples of framework regions include the following (subgroup I consensus).

[0078] a) V L FR1: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 63);

[0079] b) V L FR2: WYQQKPGKAPKLLIY (SEQ ID NO: 64);

[0080] c) V L FR3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 65); and

[0081] d) V L FR4: FGQGTKVEIK (SEQ ID NO: 66).

[0082] The V L consensus human V L framework regions suitable for use with the CDRs described herein include the following (subgroup II consensus).

[0083] a) V L FR1: DIVMTQSPLSLPVTPGEPASISC (SEQ ID NO: 67);

[0084] b) V L FR2: WYLQKPGQSPQLLIY (SEQ ID NO: 68);

[0085] c) V L FR3: GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC (SEQ ID NO: 69); and

[0086] d) V L FR4: FGQGTKVEIK (SEQ ID NO: 70).

[0087] V as described herein L Consensus human V suitable for use with CDR L Non-limiting examples of framework regions include the following (Subgroup III consensus).

[0088] a) V L FR1: DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 71);

[0089] b) V L FR2: WYQQKPGQPPKLLIY (SEQ ID NO: 72);

[0090] c) V L FR3: GVPDRFSGSGSGTDFTLTISSLQAEDFAVYYC (SEQ ID NO: 73); and

[0091] d) V L FR4: FGQGTKVEIK (SEQ ID NO: 74).

[0092] V as described herein L Consensus human V suitable for use with CDR L Non-limiting examples of framework regions include the following (Subgroup IV consensus).

[0093] a) V L FR1: DIVMTQSPDSLAVSLGERATINC (SEQ ID NO: 71);

[0094] b) V L FR2: WYQQKPGQPPKLLIY (SEQ ID NO: 72);

[0095] c) V L FR3: GVPDRFSGSGSGTDFTLTISSLQAEDFAVYYC (SEQ ID NO: 73); and

[0096] d) V LFR4: FGQGTKVEIK (SEQ ID NO:74).

[0097] In some cases, the anti-Bb antibodies of the present disclosure include a light chain variable region comprising the amino acid sequences SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0098] In some cases, the anti-Bb antibodies of the present disclosure include a heavy chain variable region comprising the amino acid sequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

[0099] In some cases, the anti-Bb antibodies of the present disclosure include a light chain variable region comprising the amino acid sequences SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0100] In some cases, the anti-Bb antibodies of the present disclosure include a heavy chain variable region comprising the amino acid sequences SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0101] In some cases, the anti-Bb antibodies of the present disclosure include a light chain variable region comprising the amino acid sequences SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0102] In some cases, the anti-Bb antibodies of the present disclosure include a heavy chain variable region comprising the amino acid sequences SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0103] In some cases, the anti-Bb antibodies of the present disclosure include a light chain variable region comprising the amino acid sequences SEQ ID NO:25, SEQ ID NO:2, and SEQ ID NO:26.

[0104] In some cases, the anti-Bb antibodies of the present disclosure include a heavy chain variable region comprising the amino acid sequences SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

[0105] In some cases, the anti-Bb antibody of the present disclosure comprises CDR-L1 having the amino acid sequence SEQ ID NO:1, CDR-L2 having the amino acid sequence SEQ ID NO:2, CDR-L3 having the amino acid sequence SEQ ID NO:3, CDR-H1 having the amino acid sequence SEQ ID NO:4, CDR-H2 having the amino acid sequence SEQ ID NO:5, and CDR-H3 having the amino acid sequence SEQ ID NO:6.

[0106] In some cases, the anti-Bb antibody of the present disclosure comprises CDR-L1 having the amino acid sequence SEQ ID NO:9, CDR-L2 having the amino acid sequence SEQ ID NO:10, CDR-L3 having the amino acid sequence SEQ ID NO:11, CDR-H1 having the amino acid sequence SEQ ID NO:12, CDR-H2 having the amino acid sequence SEQ ID NO:13, and CDR-H3 having the amino acid sequence SEQ ID NO:14.

[0107] In some cases, the anti-Bb antibody of the present disclosure comprises CDR-L1 having the amino acid sequence SEQ ID NO:17, CDR-L2 having the amino acid sequence SEQ ID NO:18, CDR-L3 having the amino acid sequence SEQ ID NO:19, CDR-H1 having the amino acid sequence SEQ ID NO:20, CDR-H2 having the amino acid sequence SEQ ID NO:21, and CDR-H3 having the amino acid sequence SEQ ID NO:22.

[0108] In some cases, the anti-Bb antibody of the present disclosure comprises CDR-L1 having the amino acid sequence SEQ ID NO:25, CDR-L2 having the amino acid sequence SEQ ID NO:2, CDR-L3 having the amino acid sequence SEQ ID NO:26, CDR-H1 having the amino acid sequence SEQ ID NO:27, CDR-H2 having the amino acid sequence SEQ ID NO:28, and CDR-H3 having the amino acid sequence SEQ ID NO:29.

[0109] In some cases, the anti-Bb antibody of this disclosure is SEQ ID NO:7, SEQ ID The amino acid sequence selected from the group consisting of NO:15, SEQ ID NO:23, and SEQ ID NO:30, and 85%, 86%, 87%, 88%, 89%, 90%, 91% , including a light chain variable region containing an amino acid sequence that is 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical.

[0110] In some cases, the anti-Bb antibodies of this disclosure include a light chain variable region containing an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence specified in SEQ ID NO:7.

[0111] In some cases, the anti-Bb antibodies of this disclosure include a light chain variable region containing an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence specified in SEQ ID NO:15.

[0112] In some cases, the anti-Bb antibodies of this disclosure include a light chain variable region containing an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence specified in SEQ ID NO:23.

[0113] In some cases, the anti-Bb antibodies of this disclosure include a light chain variable region containing an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence specified in SEQ ID NO:30.

[0114] In some cases, the anti-Bb antibody of this disclosure is SEQ ID NO:8, SEQ ID It includes a heavy chain variable region containing an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group consisting of NO:16, SEQ ID NO:24, and SEQ ID NO:31.

[0115] In some cases, the anti-Bb antibodies of this disclosure include a heavy chain variable region containing an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence specified in SEQ ID NO:8.

[0116] In some cases, the anti-Bb antibodies of this disclosure include a heavy chain variable region containing an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence specified in SEQ ID NO:16.

[0117] In some cases, the anti-Bb antibodies of this disclosure include a heavy chain variable region containing an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence specified in SEQ ID NO:24.

[0118] In some cases, the anti-Bb antibodies of this disclosure include a heavy chain variable region containing an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence specified in SEQ ID NO:31.

[0119] In some cases, the anti-Bb antibody of this disclosure has the amino acid sequence SEQ ID NO:7 and It contains a light chain variable region with an amino acid sequence that is 90% identical.

[0120] In some cases, the anti-Bb antibody of the present disclosure includes a light chain variable region comprising an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:15.

[0121] In some cases, the anti-Bb antibody of the present disclosure includes a light chain variable region comprising an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:23.

[0122] In some cases, the anti-Bb antibody of the present disclosure includes a light chain variable region comprising an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:30.

[0123] In some cases, the anti-Bb antibody of the present disclosure includes a heavy chain variable region comprising an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:8.

[0124] In some cases, the anti-Bb antibody of the present disclosure includes a heavy chain variable region comprising an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:16.

[0125] In some cases, the anti-Bb antibody of the present disclosure includes a heavy chain variable region comprising an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:24.

[0126] In some cases, the anti-Bb antibody of the present disclosure includes a heavy chain variable region comprising an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:31.

[0127] In some cases, the anti-Bb antibody of the present disclosure includes a light chain variable region comprising an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:7.

[0128] In some cases, the anti-Bb antibody of the present disclosure includes a light chain variable region comprising an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:15.

[0129] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:23.

[0130] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:30.

[0131] In some cases, the anti-Bb antibody of this disclosure includes a heavy chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:8.

[0132] In some cases, the anti-Bb antibody of this disclosure includes a heavy chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:16.

[0133] In some cases, the anti-Bb antibody of this disclosure includes a heavy chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:24.

[0134] In some cases, the anti-Bb antibody of this disclosure includes a heavy chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:31.

[0135] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing the amino acid sequence SEQ ID NO:7.

[0136] In some cases, the anti-Bb antibody of this disclosure includes a heavy chain variable region containing the amino acid sequence SEQ ID NO:8.

[0137] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing the amino acid sequence SEQ ID NO:15.

[0138] In some cases, the anti-Bb antibody of this disclosure includes a heavy chain variable region containing the amino acid sequence SEQ ID NO:16.

[0139] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing the amino acid sequence SEQ ID NO:23.

[0140] In some cases, the anti-Bb antibody of this disclosure includes a heavy chain variable region containing the amino acid sequence SEQ ID NO:24.

[0141] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing the amino acid sequence SEQ ID NO:30.

[0142] In some cases, the anti-Bb antibody of this disclosure includes a heavy chain variable region containing the amino acid sequence SEQ ID NO:31.

[0143] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:7, and a heavy chain variable region containing an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:8.

[0144] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:15, and a heavy chain variable region containing an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:16.

[0145] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:23, and a heavy chain variable region containing an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:24.

[0146] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:30, and a heavy chain variable region containing an amino acid sequence that is 90% identical to amino acid sequence SEQ ID NO:31.

[0147] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:7, and a heavy chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:8.

[0148] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:15, and a heavy chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:16.

[0149] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:23, and a heavy chain variable region containing an amino acid sequence that is 95% identical to amino acid sequence SEQ ID NO:24.

[0150] In some cases, the anti-Bb antibody of this disclosure has the amino acid sequence SEQ ID NO:30 It includes a light chain variable region containing an amino acid sequence that is 95% identical to that of SEQ ID NO:31, and a heavy chain variable region containing an amino acid sequence that is 95% identical to that of SEQ ID NO:31.

[0151] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region including amino acid sequence SEQ ID NO:7 and a heavy chain variable region including amino acid sequence SEQ ID NO:8.

[0152] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region including amino acid sequence SEQ ID NO:15 and a heavy chain variable region including amino acid sequence SEQ ID NO:16.

[0153] In some cases, the anti-Bb antibody of this disclosure comprises a light chain variable region including amino acid sequence SEQ ID NO:23 and a heavy chain variable region including amino acid sequence SEQ ID NO:24.

[0154] In some cases, the anti-Bb antibody of this disclosure includes a light chain variable region containing amino acid sequence SEQ ID NO:30 and a heavy chain variable region containing amino acid sequence SEQ ID NO:31.

[0155] In some cases, the anti-Bb antibody of this disclosure specifically binds to an epitope in the complement Bb protein, and the antibody competes for binding to the epitope with an antibody comprising a light chain CDR in the antibody light chain variable region containing amino acid sequence SEQ ID NO:7 and a heavy chain CDR in the antibody heavy chain variable region containing amino acid sequence SEQ ID NO:8.

[0156] In some cases, the anti-Bb antibody of this disclosure specifically binds to an epitope in the complement Bb protein, and the antibody competes for binding to the epitope with an antibody comprising a light chain CDR in the antibody light chain variable region containing amino acid sequence SEQ ID NO:15 and a heavy chain CDR in the antibody heavy chain variable region containing amino acid sequence SEQ ID NO:16.

[0157] In some cases, the anti-Bb antibody of this disclosure specifically binds to an epitope in the complement Bb protein, and the antibody competes for binding to the epitope with an antibody comprising a light chain CDR in the antibody light chain variable region containing amino acid sequence SEQ ID NO:23 and a heavy chain CDR in the antibody heavy chain variable region containing amino acid sequence SEQ ID NO:24.

[0158] In some cases, the anti-Bb antibody of this disclosure specifically binds to an epitope in the complement Bb protein, and the antibody competes for binding to the epitope with an antibody comprising a light chain CDR in the antibody light chain variable region containing amino acid sequence SEQ ID NO:30 and a heavy chain CDR in the antibody heavy chain variable region containing amino acid sequence SEQ ID NO:31.

[0159] In some cases, the anti-Bb antibody of this disclosure comprises a light chain CDR of the antibody light chain variable region containing amino acid sequence SEQ ID NO:7, and a heavy chain CDR of the antibody heavy chain variable region containing amino acid sequence SEQ ID NO:8.

[0160] In some cases, the anti-Bb antibody of this disclosure comprises a light chain CDR of the antibody light chain variable region containing amino acid sequence SEQ ID NO:15, and a heavy chain CDR of the antibody heavy chain variable region containing amino acid sequence SEQ ID NO:16.

[0161] In some cases, the anti-Bb antibody of this disclosure comprises a light chain CDR of the antibody light chain variable region including amino acid sequence SEQ ID NO:23, and a heavy chain CDR of the antibody heavy chain variable region including amino acid sequence SEQ ID NO:24.

[0162] In some cases, the anti-Bb antibody of this disclosure comprises a light chain CDR of the antibody light chain variable region including amino acid sequence SEQ ID NO:30, and a heavy chain CDR of the antibody heavy chain variable region including amino acid sequence SEQ ID NO:31.

[0163] In some cases, the anti-Bb antibody of this disclosure binds to complement Bb protein derived from an individual having a complement system. In some embodiments, the anti-Bb antibody of this disclosure binds to complement Bb protein derived from a mammal, fish, or invertebrate having a complement system. In some embodiments, the anti-Bb antibody of this disclosure binds to mammalian complement Bb protein. In some embodiments, the anti-Bb antibody of this disclosure binds to human complement Bb protein. In some embodiments, the anti-Bb antibody of this disclosure binds to complement Bb protein having amino acids 26-259 of the amino acid sequence depicted in Figure 13. Figure 13 provides the amino acid sequence of Homo sapiens complement Bb protein; amino acids 26-259 are the mature protein.

[0164] In some cases, the anti-Bb antibody of this disclosure is 10 -8 M~10 -9 M, 10 -9 M~10 -10 M, or 10 -10 M~10 -11 It binds to complement Bb protein with M affinity.

[0165] In some cases, the anti-Bb antibody of this disclosure exhibits preferential binding to factor Bb compared to the binding of the anti-Bb antibody to factor B. In some cases, the anti-Bb antibody of this disclosure binds to factor Bb but substantially does not bind to soluble factor B. In some cases, the anti-Bb antibody of this disclosure binds to factor Bb with an affinity at least 2 times, at least 2.5 times, at least 3 times, at least 4 times, at least 5 times, at least 7.5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, at least 75 times, or at least 100 times higher than the affinity of the antibody to factor B. In some cases, the anti-Bb antibody of the Disclosure binds to factor Bb with an affinity 2 to 2.5 times, 2.5 to 5 times, 5 to 10 times, 10 to 15 times, 15 to 20 times, 20 to 25 times, 25 to 50 times, 50 to 75 times, or 75 to 100 times higher than the affinity of the antibody to factor B. In some cases, the ratio of i) binding of the anti-Bb antibody of the Disclosure to factor Bb to ii) binding of the anti-Bb antibody of the Disclosure to factor B is at least 2:1, at least 5:1, at least 10:1, at least 25:1, at least 50:1, at least 75:1, or at least 100:1. In some cases, the ratio of i) binding of the anti-Bb antibody of this disclosure to factor Bb versus ii) binding of the anti-Bb antibody of this disclosure to factor B is 2:1–5:1, 5:1–10:1, 10:1–25:1, 25:1–50:1, 50:1–75:1, or 75:1–100:1.

[0166] In some cases, the anti-Bb antibody of this disclosure binds to both factor B and factor Bb.

[0167] In some cases, the anti-Bb antibodies of this disclosure inhibit alternative pathway (AP) activity by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the level of AP activity in the absence of the anti-Bb antibody. In some cases, the anti-Bb antibodies of this disclosure inhibit 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 activity.

[0168] In some cases, the anti-Bb antibody of this disclosure is formed in the absence of the anti-Bb antibody. Compared to the amount of MAC present, it 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%.

[0169] In some cases, the anti-Bb antibodies of this disclosure inhibit the C3b / Bb-mediated cleavage of C3. C3b / Bb is also known as "C3 convertase." In some cases, the anti-Bb antibodies of this disclosure inhibit the 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 the cleavage of C3 in the absence of the anti-Bb antibody. In some cases, the anti-Bb antibodies of this disclosure inhibit the 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.

[0170] In some cases, the anti-Bb antibodies of this disclosure inhibit C3b / Bb-mediated cleavage of C3, thereby reducing the production of C3 cleavage products. For example, in some cases, the anti-Bb antibodies of this disclosure 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 antibody. In some cases, the anti-Bb antibodies of this disclosure that inhibit C3b / Bb-mediated cleavage of C3 include VH and VL CDRs present in M17 VH and VL, respectively. In some cases, the anti-Bb antibodies of this disclosure that inhibit C3b / Bb-mediated cleavage of C3 include VH and VL CDR present in M10 VH and VL, respectively.

[0171] In some cases, the anti-Bb antibodies of this disclosure bind to and inhibit the cleavage of factor B. For example, in some cases, the anti-Bb antibodies of this disclosure inhibit factor B cleavage 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 more than 95% compared to factor B cleavage in the absence of the anti-Bb antibody. In some cases, the anti-Bb antibodies of this disclosure inhibit C3b / Bb formation.

[0172] In some cases, the anti-Bb antibodies of this disclosure inhibit complement AP-mediated cytolysis by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the degree of cytolysis in the absence of the anti-Bb antibody. The degree of inhibition of AP-mediated cytolysis can be determined using a cytolysis assay. In some cases, the anti-Bb antibodies of this disclosure inhibit 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 -8 M, 10 -8 M~5×10 -8 M, or 5×10 -8 M~10 -9 M IC 50 This inhibits AP-mediated cell lysis.

[0173] In some cases, the anti-Bb antibody of this disclosure reduces complement AP-mediated hemolysis by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, and at least 65% compared to the degree of hemolysis in the absence of the anti-Bb antibody. It inhibits %, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. The degree of inhibition of AP-mediated hemolysis can be determined using a rabbit erythrocyte (RBC) hemolysis assay. In some cases, the anti-Bb antibody of this disclosure inhibits 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 50This inhibits AP-mediated hemolysis.

[0174] In some cases, the anti-Bb antibodies of this disclosure inhibit AP-mediated deposition of C3b, C3d, or other C3 fragmentation products onto cells or tissues. For example, in some cases, the anti-Bb antibodies of this disclosure inhibit AP-mediated deposition of C3b, C3d, or other C3 fragmentation products onto 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 fragmentation products onto cells or tissues in the absence of or before administration of the anti-Bb antibody. In some cases, the anti-Bb antibodies of this disclosure inhibit AP-mediated deposition of C3b, C3d, or other C3 fragmentation products onto 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 fragmentation products onto cells or tissues in the absence of or before administration of the anti-Bb 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 resolution products onto cells or tissues.

[0175] In some cases, the anti-Bb antibodies of this disclosure inhibit 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 anti-Bb antibody. -7 M~10 -9 M IC 50 For example, 10 -7M~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 cells or tissues.

[0176] In some cases, the anti-Bb antibodies of this disclosure inhibit AP-mediated C3b deposition on red blood cells (RBCs) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the amount of C3b deposition on RBCs in the absence of the anti-Bb 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 C3b deposition on RBCs.

[0177] In some cases, the anti-Bb antibodies of this disclosure, when administered in one or more doses to an individual in need of treatment, reduce the amount of circulating Bb factor in that individual. For example, in some cases, the anti-Bb antibodies of this disclosure, when administered in one or more doses to an individual in need of treatment, reduce the amount of circulating Bb factor in that individual by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the amount of circulating Bb factor in that individual in the absence of the anti-Bb antibody administration, or compared to the amount of circulating Bb factor in that individual before the administration of the anti-Bb antibody. To make someone do it.

[0178] In some cases, the anti-Bb antibodies of this disclosure inhibit the H factor (fH) interaction with C3bBb (complement alternative pathway C3 convertase). In some cases, the anti-Bb antibodies of this disclosure inhibit the fH interaction with C3bBb 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 interaction of fH with C3bBb in the absence of the anti-Bb antibody. In some cases, the anti-Bb antibodies of this disclosure that inhibit the fH interaction with C3bBb include VH and VL CDR present in M4 VH and VL, respectively.

[0179] In some cases, the anti-Bb antibodies of this disclosure inhibit the binding of factor H (fH) to C3bBb (complement alternative pathway C3 convertase). In some cases, the anti-Bb antibodies of this disclosure inhibit the binding of fH to C3bBb 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 binding of fH to C3bBb in the absence of the anti-Bb antibody. In some cases, the anti-Bb antibodies of this disclosure that inhibit the binding of fH to C3bBb include VH and VL CDR present in M4 VH and VL, respectively.

[0180] In some cases, anti-Bb antibodies of the present disclosure that inhibit the fH interaction with C3bBb induce C3 degradation. In some cases, anti-Bb antibodies of the present disclosure that inhibit the fH interaction with C3bBb, when administered in one or more doses to an individual in need of such antibody administration, reduce the amount of C3 in the body fluids or tissues of that individual by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the amount of C3 in the body fluids or tissues in the individual in the absence of administration of the anti-Bb antibody, or compared to the amount of circulating Bb factor in the individual before administration of the anti-Bb antibody. Body fluids include, for example, serum, plasma, lymph, extracellular fluid, blood, etc.

[0181] In some cases, anti-Bb antibodies of the present disclosure that inhibit fH binding to C3bBb induce C3 degradation. In some cases, anti-Bb antibodies of the present disclosure that inhibit fH binding to C3bBb, when administered in one or more doses to an individual in need of such antibody administration, reduce the amount of C3 in the body fluids or tissues of that individual by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the amount of C3 in the body fluids or tissues in the individual in the absence of administration of the anti-Bb antibody, or compared to the amount of circulating Bb factor in the individual before administration of the anti-Bb antibody. Body fluids include, for example, serum, plasma, lymph, extracellular fluid, blood, etc.

[0182] In some cases, the anti-Bb antibodies of this disclosure that inhibit the fH interaction with C3bBb, when such antibodies are administered in one or more doses to an individual having fulminant antiphospholipid syndrome, increase the amount of C3 in the body fluids or tissues of the individual by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, and at least 8% compared to the amount of C3 in the body fluids or tissues of the individual in the absence of administration of the anti-Bb antibody, or compared to the amount of circulating Bb factor in the individual before administration of the anti-Bb antibody. Reduce by 5%, at least 90%, or at least 95%.

[0183] In some cases, anti-Bb antibodies of the present disclosure that inhibit fH binding to C3bBb reduce the amount of C3 in the body fluids or tissues of an individual having fulminant antiphospholipid syndrome 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 C3 in the body fluids or tissues of the individual in the absence of administration of the anti-Bb antibody, or compared to the amount of circulating Bb factor in the individual before administration of the anti-Bb antibody.

[0184] This disclosure provides any embodiment of anti-Bb antibodies that are subject to humanization. In some cases, the anti-Bb antibodies of this disclosure include a humanized framework region. In some cases, the anti-Bb antibodies of this disclosure include a humanized light chain framework region. In some cases, the anti-Bb antibodies of this disclosure include a humanized heavy chain framework region. In some cases, the anti-Bb antibodies of this disclosure include both a humanized light chain framework region and a humanized heavy chain framework region.

[0185] In some cases, the anti-Bb antibody in question includes one or more humanized framework regions (FRs). In some cases, the anti-Bb antibody in question includes a light chain variable region comprising one, two, three, or four humanized light chain FRs. In some embodiments, the anti-Bb antibody in question includes a light chain variable region comprising, in the order from N-terminus to C-terminus: humanized light chain FR1; CDR-L1 as specified herein; humanized light chain FR2; CDR-L2 as specified herein; humanized light chain FR3; CDR-L3 as specified herein; and humanized light chain FR4. In some cases, the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are one of the following combinations: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; and SEQ ID NO:25, SEQ ID NO:2, and SEQ ID NO:26.

[0186] For example, the anti-Bb antibody of this disclosure may include a light chain variable region comprising, in order from N-terminus to C-terminus: humanized light chain FR1; CDR-L1 containing amino acid sequence SEQ ID NO:1; humanized light chain FR2; CDR-L2 containing amino acid sequence SEQ ID NO:2; humanized light chain FR3; CDR-L3 containing amino acid sequence SEQ ID NO:3; and humanized light chain FR4.

[0187] In some cases, the anti-Bb antibodies of this disclosure include a heavy chain variable region comprising one, two, three, or four humanized heavy chain FRs. In some cases, the antibody in question includes a heavy chain variable region comprising, in N-terminal to C-terminal order: humanized heavy chain FR1; CDR-H1 as specified herein; humanized heavy chain FR2; CDR-H2 as specified herein; humanized heavy chain FR3; CDR-H3 as specified herein; and humanized heavy chain FR4. For example, the antibody in question may include a heavy chain variable region comprising, in N-terminal to C-terminal order: humanized heavy chain FR1; CDR-H1 with amino acid sequence SEQ ID NO: 4; humanized heavy chain FR2; CDR-H2 with amino acid sequence SEQ ID NO: 5; humanized heavy chain FR3; CDR-H3 with amino acid sequence SEQ ID NO: 6; and humanized heavy chain FR4. In some embodiments, the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are in the following combinations: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; SEQ ID NO:12, SEQ ID NO:13, And one of SEQ ID NO:14; SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; and SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

[0188] For example, the anti-Bb antibody of this disclosure may include a heavy chain variable region comprising, in order from N-terminus to C-terminus: humanized heavy chain FR1; CDR-L1 containing amino acid sequence SEQ ID NO:4; humanized heavy chain FR2; CDR-L2 containing amino acid sequence SEQ ID NO:5; humanized heavy chain FR3; CDR-L3 containing amino acid sequence SEQ ID NO:6; and humanized heavy chain FR4.

[0189] In some embodiments, the anti-Bb antibody of this disclosure that binds to human complement Bb protein also binds to complement Bb protein of another species. In some embodiments, the anti-Bb antibody of this disclosure that binds to human complement Bb protein also binds to non-human primate complement Bb protein. In some embodiments, the anti-Bb antibody of this disclosure that binds to human complement Bb protein also binds to rodent complement Bb protein. Examples of rodent complement Bb proteins include, but are not limited to, guinea pig Bb protein, hamster Bb protein, mouse Bb protein, and rat Bb protein. In some embodiments, such cross-reactive antibodies bind to human complement Bb protein at a similar order of magnitude as the antibody binds to human complement Bb protein. D Then, it binds to the complement Bb protein of another species.

[0190] In some cases, the anti-Bb antibody of this disclosure is an Ig monomer or an antigen-binding fragment thereof that binds to the complement Bb protein. In some cases, the anti-Bb antibody of this disclosure is an Ig monomer. In some cases, the anti-Bb antibody of this disclosure is an antigen-binding fragment of an Ig monomer that binds to the complement Bb protein.

[0191] In some cases, the anti-Bb antibodies of this disclosure are selected from the group consisting of Ig monomers, Fab fragments, F(ab')2 fragments, Fd fragments, scFv, scAb, dAb, Fv, single-domain heavy chain antibodies, and single-domain light chain antibodies. In some cases, the anti-Bb antibodies of this disclosure are single-chain Fv(scFv) antibodies.

[0192] In some cases, the anti-Bb antibodies of this disclosure include a light chain region and a heavy chain region that are present in separate polypeptides.

[0193] In some cases, the anti-Bb antibodies of this disclosure include a light chain region and a heavy chain region present in a single polypeptide.

[0194] In some embodiments, the anti-Bb antibody of the Disclosure comprises an anti-Bb heavy chain CDR and an anti-Bb light chain CDR in a single polypeptide chain, for example, in some embodiments, the antibody of interest is scFv. In some embodiments, the anti-Bb antibody of the Disclosure comprises, in order from N-terminal to C-terminal: a first amino acid sequence of about 5 to 25 amino acids in length; CDR-L1; a second amino acid sequence of about 5 to 25 amino acids in length; CDR-L2; a third amino acid sequence of about 5 to 25 amino acids in length; CDR-L3; a fourth amino acid sequence of about 5 to 25 amino acids in length; CDR-H1; a fifth amino acid sequence of about 5 to 25 amino acids in length; CDR-H2; a sixth amino acid sequence of about 5 to 25 amino acids in length; CDR-H3; and a seventh amino acid sequence of about 5 to 25 amino acids in length. In some cases, the amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are in the following combinations: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N O:12, SEQ ID NO:13, and SEQ ID NO:14; SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; and SEQ It is one of ID NO:25, SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29. For example, in some cases, the anti-Bb antibody of this disclosure is in the order from N-terminus to C-terminus: a first amino acid sequence of approximately 5 to 25 amino acids in length; CDR-L1 containing the amino acid sequence specified in SEQ ID NO:1; a second amino acid sequence of approximately 5 to 25 amino acids in length; CDR-L2 containing the amino acid sequence specified in SEQ ID NO:2; a third amino acid sequence of approximately 5 to 25 amino acids in length; CDR-L3 containing the amino acid sequence specified in SEQ ID NO:3; a fourth amino acid sequence of approximately 5 to 25 amino acids in length; CDR-H1 containing the amino acid sequence specified in SEQ ID NO:4; a fifth amino acid sequence of approximately 5 to 25 amino acids in length; SEQ ID It includes CDR-H2 containing the amino acid sequence specified in NO:5; a sixth amino acid sequence with a length of approximately 5 to 25 amino acids; CDR-H3 containing the amino acid sequence specified in SEQ ID NO:6; and a seventh amino acid sequence with a length of approximately 5 to 25 amino acids.

[0195] In some embodiments, the anti-Bb antibody of the present disclosure comprises, in the order from N-terminus to C-terminus: light chain FR1 region; CDR-L1; light chain FR2 region; CDR-L2; light chain FR3 region; CDR-L3; optionally light chain FR4 region; linker region; optionally heavy chain FR1 region; CDR-H1; heavy chain FR2 region; CDR-H2; heavy chain FR3 region; CDR-H3; and heavy chain FR4 region. In some embodiments, the amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are in the following combinations: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; and SEQ ID NO:25, SEQ ID NO:2, SEQ ID NO:26, SEQ ID One of NO:27, SEQ ID NO:28, and SEQ ID NO:29. In some of these embodiments, one or more FR regions are humanized FR regions. In some of these embodiments, each of the FR regions is a humanized FR region. The linker region may have a length of about 5 amino acids (aa) to about 50 amino acids, for example, about 5aa to about 10aa, about 10aa to about 15aa, about 15aa to about 20aa, about 20aa to about 25aa, about 25aa to about 30aa, about 30aa to about 35aa, about 35aa to about 40aa, about 40aa to about 45aa, or about 45aa to about 50aa.

[0196] In some cases, the anti-Bb antibody of this disclosure comprises, in the order from N-terminus to C-terminus: heavy chain FR1 region; CDR-H1; heavy chain FR2 region; CDR-H2; heavy chain FR3 region; CDR-H3; optionally heavy chain FR4 region; linker; optionally light chain FR1 region; CDR-L1; light chain FR2 region; CDR-L2; light chain FR3 region; CDR-L3; and light chain FR4 region. In some embodiments, the amino acid sequences of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 are the following combinations: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22; and one of SEQ ID NO:25, SEQ ID NO:2, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29. In some of these embodiments, one or more FR regions are humanized FR regions. In some of these embodiments, each of the FR regions is a humanized FR region. The linker region may be about 5 to 50 amino acids in length, for example, about 5aa to 10aa, about 10aa to 15aa, about 15aa to 20aa, about 20aa to 25aa, about 25aa to 30aa, about 30aa to 35aa, about 35aa to 40aa, about 40aa to 45aa, or about 45aa to 50aa.

[0197] Appropriate linkers for use with the target antibody include “flexible linkers.” If present, the linker molecule is generally of sufficient length to allow some flexible movement between linked regions. In some embodiments, the linker molecule is typically about 6–50 atomic lengths. The linker molecule may also be, for example, arylacetylene, ethylene glycol oligomers containing 2–10 monomer units, diamines, diacitors, amino acids, or combinations thereof. Other linker molecules capable of binding to polypeptides may be used in light of this disclosure.

[0198] A suitable linker can be easily selected and may be of any of several suitable lengths, including 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 to 10 amino acids, 5 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0199] An example of a flexible linker is glycine polymer (G) n , Glycine-serine polymer (e.g., (GS) n (GSGGS) n (SEQ ID NO:75), and (GGGS) nThe formula includes (SEQ ID NO: 76), where n is at least an integer of 1, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest because both of these amino acids are relatively unstructured and can therefore act as neutral constraints between components. Glycine polymers are of particular interest because glycine is significantly closer to the phi-psi space than even alanine and is far less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Examples of flexible linkers include, but are not limited to, GGSG (SEQ ID NO: 77), GGSGG (SEQ ID NO: 78), GSGSG (SEQ ID NO: 79), GSGGG (SEQ ID NO: 80), GGGSG (SEQ ID NO: 81), GSSSG (SEQ ID NO: 82), etc. Those skilled in the art will recognize that the design of a peptide conjugated to any of the elements described above may include a linker that is fully or partially flexible, thereby including one or more parts that confer both a flexible linker and a less flexible structure.

[0200] In some embodiments, the anti-Bb antibody of the Disclosure comprises scFv polymers. For example, in some embodiments, the anti-Bb antibody of the Disclosure is an scFv dimer (e.g., comprising two tandem scFv(scFv2)), an scFv trimer (e.g., comprising three tandem scFv(scFv3)), an scFv tetramer (e.g., comprising four tandem scFv(scFv4)), or a polymer of more than four scFv (e.g., in tandem). The scFv monomers may be linked in tandem via linkers of length from about 2 to about 10 amino acids (aa), for example, 2aa, 3aa, 4aa, 5aa, 6aa, 7aa, 8aa, 9aa, or 10aa. Suitable linkers include, for example, (Gly) x The formula includes, and in the formula, x is The integers are between 2 and 10. Other suitable linkers are those described above. In some embodiments, as described above, each of the scFv monomers in the scFv polymer of interest is humanized.

[0201] In some cases, the anti-Bb antibodies of this disclosure include a constant region of the immunoglobulin (e.g., an Fc region). The Fc region may be a human Fc region, if present, or an Fc region derived from any animal having a complement system. In some embodiments, the Fc region is a human Fc region, if present. In some cases, the Fc region includes one or more mutations (e.g., amino acid substitutions) that increase the affinity of Fc to the neonatal Fc receptor (FcRn); see, for example, Monnet et al. (2015) Front.Immunol. 6:39. Examples of amino acid substitutions that increase the affinity of the Fc polypeptide to FcRn include, for example, the combination of M428L and N434S; and the combination of M252Y, S254T, and T256E. If a constant region is present, the antibody may contain both light-chain and heavy-chain constant regions. Suitable heavy-chain constant regions include the CH1, hinge, CH2, CH3, and CH4 regions. The antibodies described herein include antibodies having all types of constant regions, including IgM, IgG, IgD, IgA, and IgE, as well as any isotype including IgG1, IgG2, IgG3, and IgG4. An example of a suitable heavy chain Fc region is human isotype IgG1 It is Fc. Another example of a suitable heavy chain Fc region is human isotype IgG2a Fc. Yet another example of a suitable heavy chain Fc region is human isotype IgG2b Fc. The light chain constant region may be lambda or kappa. The antibody of interest (e.g., the humanized antibody of interest) may contain sequences derived from more than one class or isotype. The antibody may be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy chains and light chains, as Fab, Fab', F(ab')2, and Fv, or as a single-chain antibody in which the heavy chain and light chain variable domains are linked through a spacer.

[0202] In some cases, the heavy chain region is of isotype IgG4. In some of these embodiments, the hinge region includes the S241P substitution. See, for example, Angal et al. (1993) Mol.Immunol. 30:105. In some of these embodiments, the hinge region includes the L236E substitution. See, for example, Reddy et al. (2000) J.Immunol. 164:1925; and Klechevsky See et al. (2010) Blood 116:1685. In some of these embodiments, the hinge region includes S241P substitution and L236E substitution.

[0203] The antibody in question may contain a free thiol (-SH) group at its carboxyl terminus, and this free thiol group can be used to attach the antibody to a second polypeptide (for example, another antibody including the antibody in question), a scaffold, a carrier, etc.

[0204] In some embodiments, the antibody of interest contains one or more amino acids that do not exist naturally. In some embodiments, the amino acids that do not exist naturally include carbonyl, acetyl, aminooxy, hydrazine, hydrazide, semicarbazide, azide, or alkyne groups. For suitable amino acids that do not exist naturally, see, for example, U.S. Patent No. 7,632,924. The encapsulation of amino acids that do not exist naturally may provide linkage to polymers, secondary polypeptides, scaffolds, etc. For example, an antibody of interest linked to a water-soluble polymer may be produced by reacting the antibody with a water-soluble polymer containing a carbonyl group (e.g., PEG), and the antibody contains an aminooxy, hydrazine, hydrazide, or semicarbazide group that does not exist naturally. As another example, an antibody of interest linked to a water-soluble polymer may be produced by reacting an antibody of interest containing an alkyne-containing amino acid with a water-soluble polymer containing an azide moiety (e.g., PEG); in some cases, the azide or alkyne group is linked to the PEG via amide linkage. It is linked to the offspring. “Naturally unencoded amino acids” refers to amino acids that are not one of the 20 common amino acids, or pyrrolicin or selenocysteine. Other terms that may be used synonymously with “naturally unencoded amino acids” are “non-natural amino acids,” “unnatural amino acids,” “amino acids that do not exist naturally,” and their various hyphenated and unhyphenated versions. The term “naturally unencoded amino acids” also includes, but is not limited to, amino acids that result from modifications (e.g., post-translational modifications) of naturally encoded amino acids (including, but not limited to, the 20 common amino acids or pyrrolicin and selenocysteine), but which themselves are not naturally incorporated into the growing polypeptide chain by the translation complex. Examples of such naturally unencoded amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

[0205] In some embodiments, the antibody of interest 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 include, for example, substituted or unsubstituted linear or branched polyalkylene, polyalkene, or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homo or heteropolysaccharides.Suitable polymers include, for example, ethylene vinyl alcohol copolymer (commonly known by the generic name EVOH or the trade name EVAL); polybutyl methacrylate; poly(hydroxyvalerate); poly(L-lactic acid); polycaprolactone; poly(lactide-co-glycolide); poly(hydroxybutyrate); poly(hydroxybutyrate-covalerate); polydioxanone; polyorthoester; polyanhydride; poly(glycolic acid); poly(D,L-lactic acid); poly(glycolic acid-co-trimethylene carbonate); polyphosphate Phosphorus esters; polyphosphoester polyurethanes; poly(amino acids); cyanoacrylates; poly(trimethylene carbonate); poly(iminocarbonates); copolymers (ether-esters) (e.g., poly(ethylene oxide)-poly(lactic acid) (PEO / PLA) copolymers); polyalkylene oxalates; polyphosphazenes; biomolecules such as fibrin, fibrinogen, cellulose, starch, collagen, and hyaluronic acid; polyurethanes; silicones; polyesters; polyolefins; polyisobutylenes and ethylene-alphaolefins This includes vinyl copolymers; acrylic polymers and copolymers; halogenated vinyl polymers and copolymers such as polyvinyl chloride; polyvinyl methyl ethers such as polyvinyl ethers; halogenated polyvinylidene such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketones such as polystyrene; polyvinyl aromatics such as polyvinyl acetate; polyvinyl esters such as ethylene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, ABS resin, and ethylene-vinyl acetate copolymer; copolymers of vinyl monomers with each other and with olefins; nylon 66 and polycaprolactam such as polyamides; alkyd resins; polycarbonates; polyoxymethylene; polyimides; polyethers; epoxy resins; polyurethanes; rayon; rayon triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate; cellophane; cellulose nitrate; cellulose propionate; cellulose ethers; amorphous Teflon®; poly(ethylene glycol); and carboxymethylcellulose.

[0206] Suitable synthetic polymers include unsubstituted and substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), and their derivatives, as well as substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) and their derivatives. Suitable naturally occurring polymers include, for example, albumin, amylose, dextran, glycogen, and their derivatives.

[0207] 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. For example, in some embodiments, if the antibody of interest contains a poly(ethylene glycol) (PEG) or methoxypoly(ethylene glycol) polymer, the PEG or methoxypoly(ethylene glycol) polymer may have a molecular weight in the range of about 0.5 kilodaltons (kDa) to 1 kDa, about 1 kDa to 5 kDa, 5 kDa to 10 kDa, 10 kDa to 25 kDa, 25 kDa to 40 kDa, or 40 kDa to 60 kDa.

[0208] As described above, in some embodiments, the antibody of interest is covalently linked to a non-peptidic synthetic polymer. In some embodiments, the antibody of interest is covalently linked to a PEG polymer. In some embodiments, the scFv polymer of interest is covalently linked to a PEG polymer. See, for example, Albrecht et al. (2006) J.Immunol.Methods 310:100. Suitable methods and reagents for protein PEGylation are well known in the art and can be found, for example, in U.S. Patent No. 5,849,860. Suitable PEGs for protein conjugation are generally soluble in water at room temperature and have the general formula R(O-CH2-CH2) n The formula contains OR, where R is a hydrogen atom or a protecting group such as an alkyl or alkanol group, and where n is an integer from 1 to 1,000. If R is a protecting group, it generally has 1 to 8 carbon atoms.

[0209] In some embodiments, the PEG conjugated to the antibody of interest is linear. In some embodiments, the PEG conjugated to the antibody of interest is branched. Examples include branched PEG derivatives such as those described in U.S. Patent No. 5,643,575, "star-PEG" and multi-armed PEG, such as those described in Shearwater Polymers, Inc.'s catalog "Polyethylene Glycol Derivatives 1997-1998". Star-PEG has been described in the art, for example, in U.S. Patent No. 6,046,305.

[0210] The antibody in question may be glycosylated; for example, it may contain covalently linked carbohydrate or polysaccharide moieties. Antibody glycosylation is typically either N-linked or O-linked. N-linking refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for the enzymatic attachment of carbohydrate moieties to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the carbohydrates N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0211] The addition of glycosylation sites to antibodies is conveniently achieved by modifying the amino acid sequence so that it contains one or more of the tripeptide sequences described above (with respect to N-linked glycosylation sites). Modification can also be made by adding or substituting one or more serine or threonine residues into the original antibody sequence (with respect to O-linked glycosylation sites). Similarly, the removal of glycosylation sites can be achieved by modifying amino acids within the native glycosylation sites of antibodies.

[0212] The antibodies in question, in some embodiments, include "radiopaque" labels, such as labels that can be easily visualized 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, including organic bismuth derivatives (e.g., U.S. Patent). This includes, but is not limited to, those listed above (see Patent No. 5,939,045), radiopaque multiurethanes (see Patent No. 5,346,981), organic bismuth composites (see, for example, Patent No. 5,256,334), and radiopaque barium polymer complexes (see, for example, Patent No. 4,866,132).

[0213] The target antibody can be covalently linked to a second moiety (e.g., lipids, polypeptides other than the target antibody, synthetic polymers, carbohydrates, etc.) using, for example, glutaraldehyde, homobifunctional crosslinkers, or heterobifunctional crosslinkers. Glutaraldehyde crosslinks polypeptides via their amino moieties. Homobifunctional crosslinkers (e.g., homobifunctional imide esters, homobifunctional N-hydroxysuccinimidyl (NHS) esters, or homobifunctional sulfhydryl reactive crosslinkers) contain two or more identical reactive moieties and can be used in a one-step reaction procedure in which the crosslinker is added to a solution containing a mixture of polypeptides to be linked. Homobifunctional NHS esters and imide esters crosslink amine-containing polypeptides. At a weakly alkaline pH, imide esters react only with primary amines to form imidoamides, and the overall charge of the crosslinked polypeptide remains unaffected. Homobifunctional sulfhydryl reactive crosslinking agents include bismaleimidehexane (BMH), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and 1,4-di-(3',2'-pyridyldithio)propionamidobutane (DPDPB).

[0214] Heterobifunctional crosslinkers have two or more different reactive moieties (e.g., an amine-reactive moiety and a sulfhydryl-reactive moiety) and crosslink one polypeptide via the amine or sulfhydryl-reactive moiety, and then react with the other polypeptide via the unreacted moiety. Numerous heterobifunctional haloacetyl crosslinkers are available, as are pyridyl disulfide crosslinkers. Carbodiimides are a typical example of heterobifunctional crosslinking reagents for coupling carboxyls to amines, resulting in an amide bond.

[0215] The antibody of interest can be immobilized on a solid support. Suitable supports are well known in the art and include, in particular, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon tips and surfaces, nitrocellulose strips, nylon membranes, sheets, duracyte, wells of reaction trays (e.g., multiwell plates), plastic tubes, etc. The solid support 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 the antibody of interest on a solid support are well known and include, but are not limited to, ionic, hydrophobic, and covalent interactions. The solid support may be soluble or insoluble, for example, in aqueous solution. In some embodiments, a suitable solid support is generally insoluble in aqueous solution.

[0216] The antibody in question includes a detectable label in some embodiments. A suitable detectable label includes any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Suitable examples include magnetic beads (e.g., Dynabeads®), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas Red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), and radiolabels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, and other enzymes commonly used in enzyme-linked immunosorbent assays (ELISA)). This includes, but is not limited to, colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

[0217] In some embodiments, the antibody in question comprises a contrast agent or radioisotope, which is suitable for use in imaging, for example, in imaging procedures performed on humans. Non-limiting examples of labeling include: 1231 I (iodine), 18F (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) is also usable and appropriate for imaging procedures in non-human mammals. The antibody of interest can be labeled using standard techniques. For example, the antibody of interest can be iodized with chloramine T or 1,3,4,6-tetrachloro-3α,6α-diphenylglycouryl. Regarding fluorination, fluorine is added to the antibody of interest during synthesis by fluoride ion substitution reaction. For reviews on the synthesis of proteins with such radioisotopes, see Muller-Gartner, H., TIB Tech., 16:122-130 (1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst., 16(2):209-244 (1999). The antibody of interest can also be labeled with contrast agents using standard techniques. For example, the antibody in question can be labeled with Gd by conjugating it with a low-molecular-weight Gd chelate such as Gd-diethylenetriaminepentaacetic acid (GdDTPA) or Gd-tetraazacyclododecanetetraacetic acid (GdDOTA). See Caravan et al., Chem. Rev. 99:2293-2352 (1999) and Lauffer et al., J. Magn. Reson. Imaging, 3:11-16 (1985). The antibody in question can also be labeled with Gd by conjugating it with, for example, a polylysine-Gd chelate. See, for example, Curtet et al., Invest. Radiol., 33(10):752-761 (1998). Alternatively, the antibody in question can be labeled with Gd by incubating avidin and a biotinylated antibody with paramagnetic polymerized liposomes containing a Gd chelating lipid. For example, see Sipkins et al., Nature Med., 4:623-626 (1998).

[0218] 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, and mK Examples of fluorescent proteins include, but are not limited to, phycobiliproteins and phycobiliprotein conjugates, including O, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, maple protein and kindling protein, B-phycoerythrin, R-phycoerythrin, and allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, and mPlum (Shaner et al. (2005) Nat. Methods 2:905-909). For example, Matz As described in et al. (1999) Nature Biotechnol. 17:969-973, any of the diverse fluorescent and colored proteins derived from anthozoan species can be used. It is suitable for use.

[0219] In some embodiments, the antibody of interest is conjugated with a therapeutic agent. An antibody-agent conjugate can be formed using any of the antibodies of interest disclosed herein. The agent may be attached to the N-terminus of the light chain, the C-terminus of the light chain, the N-terminus of the heavy chain, or the C-terminus of the heavy chain. In some embodiments, the agent may be attached to the hinge of the antibody or to one or more other sites of the antibody. With respect to single-chain antibodies, the agent may be attached to the N or C-terminus of the single-chain antibody. The agent may be conjugated to the antibody 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.

[0220] In some embodiments, the target antibody is linked to a fusion partner, such as a ligand; epitope tag; peptide; or protein other than the antibody (e.g., covalently or noncovalently). Suitable fusion partners include peptides and polypeptides that confer enhanced in vivo stability (e.g., enhanced serum half-life); and peptides and polypeptides that provide ease of purification, such as (His) n , e.g., 6His; peptides and polypeptides that provide secretion of fusion proteins from cells; peptides and polypeptides that provide epitope tags, e.g., GST, hemagglutinin (HA; e.g., YPYDVPDYA; SEQ ID NO: 83), FLAG (e.g., DYKDDDDK; SEQ This includes ID NO:84), c-myc (e.g., EQKLISEEDL; SEQ ID NO:85), etc.; peptides and polypeptides that provide detectable signals, such as enzymes that produce detectable products (e.g., β-galactosidase, luciferase), or proteins that are themselves detectable, such as green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.; peptides and polypeptides that provide polymerization, such as polymerizing domains such as the Fc portion of immunoglobulins; etc.

[0221] The fusion may also include an affinity domain containing a peptide sequence that can interact with a binding partner useful for identification or purification, such as one immobilized on a solid support. Consecutive single amino acids, such as histidine, when fused to a protein, can be used for one-step purification of the fusion protein by high-affinity binding to a resin column such as nickel Sepharose. Exemplary affinity domains include His5 (HHHHH) (SEQ ID NO: 86), His×6 (HHHHHH) (SEQ ID NO: 87), C-myc (EQKLISEEDL) (SEQ ID NO: 88), Flag (DYKDDDDK) (SEQ ID NO: 85), StrepTag (WSHPQFEK) (SEQ ID NO: 89), hemagglutinin, e.g., HA tag (YPYDVPDYA; SEQ ID NO: 90), glutathione-S-transferase (GST), thioredoxin, cellulose-binding domain, RYIRS (SEQ ID NO: 91), Phe-His-His-Thr (SEQ ID NO: 92), chitin-binding domain, S-peptide, T7 peptide, SH2 domain, C-terminal RNA tag, WEAAAREACCRECCARA (SEQ ID NO:93) These include metal-binding domains, such as zinc-binding domains, or calcium-binding proteins, such as calmodulin, troponin C, calcineurin B, myosin light chain, recoverin, S-modulin, vidinin, VILIP, neurocalcin, hypocalcin, phryquenin, caltractin, calpain large subunit, S100 protein, parvalbumin, calbindin D9K, calbindin D28K, and calcium-binding domains derived from calretinin, intein, biotin, streptavidin, MyoD, leucine zipper sequences, and maltose-binding proteins.

[0222] In some embodiments, the anti-Bb antibody of this disclosure is formulated with an agent that promotes crossing of the blood-brain barrier (BBB). In some embodiments, the antibody promotes crossing of the BBB. The anti-Bb antibody is fused to a compound either directly or via a linker. Examples of such compounds include, but are not limited to, carrier molecules, peptides, or proteins. In some embodiments, the anti-Bb antibody of this disclosure is fused to a polypeptide that binds to an endogenous BBB receptor. Linking the anti-Bb antibody of this disclosure to a polypeptide that binds to an endogenous BBB receptor facilitates BBB crossing, for example, in targeted therapies involving the administration of the anti-Bb antibody of this disclosure to an individual requiring administration (see below). Suitable polypeptides that bind to an endogenous BBB receptor include antibodies that specifically bind to an endogenous BBB receptor, such as monoclonal antibodies or their antigen-binding fragments. Suitable endogenous BBB receptors include, but are not limited to, insulin receptors, transferrin receptors, leptin receptors, lipoprotein receptors, and insulin-like growth factor receptors. See, for example, U.S. Patent Publication No. 2009 / 0156498.

[0223] For example, the anti-Bb antibody in question 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 the endogenous BBB receptor.

[0224] For example, the anti-Bb antibody of this disclosure may be fused to a peptide that promotes crossing of the BBB, the peptide having a length of approximately 15 to 25 amino acids, and the following peptides: Angiopep-1 (TFFYGGCRGKRNNFKTEEY) (SEQ ID NO: 93); Angiopep-2 (TFFYGGSRGKRNNFKTEEY) (SEQ ID NO: 93); The amino acid sequence contains at least approximately 85% amino acid sequence identity with one of the following: NO:94);cys-Angiopep-2(CTFFYGGSRGKRNNFKTEEY)(SEQ ID NO:95);Angiopep-2-cys(TFFYGGSRGKRNNFKTEEYC)(SEQ ID NO:96);and aprotinin fragment(TFVYGGCRAKRNNFKS)(SEQ ID NO:97). See, for example, U.S. Patent Publication Nos. 2011 / 0288011 and 2009 / 0016959. The BBB-crossing peptide can be fused to the N-terminus of an anti-Bb light chain region, the C-terminus of an anti-Bb light chain region, the N-terminus of an anti-Bb heavy chain region, the C-terminus of an anti-Bb heavy chain region, the N-terminus of a target anti-Bb single-chain antibody, the C-terminus of a target anti-Bb single-chain antibody, etc.

[0225] In some embodiments, the antibody of interest includes polyamine modification. Polyamine modification of the antibody of interest enhances the permeability of the modified antibody at the blood-brain barrier (BBB). The antibody of interest may be modified with naturally occurring or synthetic polyamines. See, for example, U.S. Patent No. 5,670,477. Useful naturally occurring polyamines include putrescine, spermidine, spermine, 1,3-diaminopropane, norspermidine, syn-homospermidine, thermine, thermospermine, cardopentamine, homocardopentamine, and canavalmine. Putrescine, spermidine, and spermine are particularly useful. Synthetic polyamines are empirically defined by formula C X H Y N Z Polyamines can be cyclic or acyclic, branched or unbranched, 3 to 12 carbon-carbon carbohydrate chains, comprising 1 to 6 NR or N(R)2 moieties, and further containing 1 to 6 NR or N(R)2 moieties, where R is H, (C1-C4) alkyl, phenyl, or benzyl. Polyamines can be linked to antibodies using any standard crosslinking method.

[0226] In some embodiments, the antibody of interest is modified to include a carbohydrate moiety, which can be covalently linked to the antibody. In some embodiments, the antibody of interest is modified to include a lipid moiety, which can be covalently linked to the antibody. Suitable lipid moieties include, for example, N-fatty acid groups such as N-lauroyl and N-oleoyl; and dode This includes fatty amines such as silamines and oleylamines; C3-C16 long-chain aliphatic lipids; etc. See, for example, U.S. Patent No. 6,638,513. In some embodiments, the antibody of interest is incorporated into liposomes (e.g., encapsulated).

[0227] Method for producing the target antibody The antibody in question (the anti-Bb antibody of this disclosure) can be produced by any known method, such as conventional synthesis methods for protein synthesis; recombinant DNA methods; etc. In some embodiments, the antibody in question is produced by a method selected from the group consisting of recombinant production and chemical synthesis.

[0228] If the target antibody is a single-chain polypeptide, it can be synthesized using standard chemical peptide synthesis techniques. When polypeptides are chemically synthesized, the synthesis can proceed in either a liquid or solid phase. Solid-phase polypeptide synthesis (SPPS), in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by the sequential addition of the remaining amino acids in the sequence, is one example of a suitable method for the chemical synthesis of the target antibody. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing the target antibody. Techniques for solid-phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963); Stewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984); and Ganesan A. 2006 Mini Rev. Med Chem. 6: 3-10 and Camarero JA et al. 2005 Protein Pept Lett. 12: 723-8. Briefly, small insoluble porous beads are treated with a functional unit on which peptide chains are constructed. After repeated coupling / deprotection cycles, the free N-terminal amines on the attached solid phase are coupled to a single N-protected amino acid unit. This unit is then deprotected, exposing it to a new N-terminal amine to which further amino acids can be attached. The peptide remains immobilized on the solid phase and undergoes a filtration step before being cleaved and detached.

[0229] Standard recombination methods can be used to produce the target antibody. For example, nucleic acids encoding light chain and heavy chain variable regions, optionally ligated to a constant region, are inserted into the expression vector. The light and heavy chains can be cloned into the same or different expression vectors. The DNA segment encoding the immunoglobulin chain is operatively ligated to a regulatory sequence in the expression vector(s) to ensure the expression of the immunoglobulin polypeptide. The regulatory sequence includes, but is not limited to, promoters (e.g., native or heterologous promoters), signal sequences, enhancer elements, repressor elements, and transcription termination sequences. The regulatory sequence may be a eukaryotic promoter system in a vector that can transform or transfect eukaryotic host cells (e.g., COS or CHO cells). Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high levels of nucleotide sequence expression, as well as antibody recovery and purification.

[0230] Due to coding degeneracy, a diverse range of nucleic acid sequences can encode each immunoglobulin amino acid sequence. The desired nucleic acid sequence can be produced by de novo solid-phase DNA synthesis or by polymerase chain reaction (PCR) mutagenesis of a variant prepared prior to that of the desired polynucleotide. Oligonucleotide-mediated mutagenesis is one example of a suitable method for preparing substitution, deletion, and insertion variants of target polypeptide DNA. (Adelman e) See t al., DNA 2:183 (1983). Briefly, the target polypeptide DNA is modified by hybridizing a single-stranded DNA template with an oligonucleotide encoding the desired mutation. After hybridization, DNA polymerase is used to synthesize the entire second complementary strand of the template, incorporating the oligonucleotide primer and encoding the selected modification in the target polypeptide DNA.

[0231] Appropriate expression vectors are typically replicable in the host organism, either as episomes or as an integral part of the host chromosomal DNA. Generally, expression vectors contain a selection marker (e.g., ampicillin resistance, hygromycin resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance) that allows for the detection of such cells transformed with the desired DNA sequence.

[0232] Escherichia coli is an example of a prokaryotic host cell that can be used to clone polynucleotides encoding target antibodies. Other suitable microbial hosts include Bacillus species such as Bacillus subtilis, as well as other enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species. Expression vectors typically containing expression regulatory sequences (e.g., origins of replication) compatible with these prokaryotic hosts can also be constructed. In addition, there are many diverse and well-known promoters, such as lactose promoter systems, tryptophan (trp) promoter systems, beta-lactamase promoter systems, or lambda phage-derived promoter systems. Promoters typically contain ribosome-binding site sequences, along with operator sequences, to regulate expression and initiate and complete transcription and translation.

[0233] Other microorganisms, such as yeast, are also useful for expression. Saccharomyces (e.g., S. cerevisiae) and Pichia are examples of suitable yeast host cells, along with appropriate vectors containing the desired expression regulatory sequences (e.g., promoters), origins of replication, termination sequences, etc. Typical promoters include 3-phosphoglycerate kinase and other glycosphagocytes. Inducible yeast promoters include, among others, those derived from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization.

[0234] In addition to microorganisms, mammalian cells (e.g., mammalian cells grown in in vitro cell culture) can also be used to express and produce the anti-Bb antibody of this disclosure (e.g., a polynucleotide encoding the target anti-Bb antibody). See Clones, VCH Publishers, NY, NY (1987). Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B cells or hybridomas. Expression vectors for these cells may contain expression regulatory sequences such as origins of replication, promoters, and enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), as well as necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences. Examples of suitable expression regulatory sequences are promoters derived from immunoglobulin genes, SV40, adenoviruses, bovine papillomavirus, cytomegalovirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0235] Once synthesized (either chemically or recombinantly), the entire antibody, its dimers, individual light and heavy chains, or other forms of the antibody of interest (e.g., scFv) can be purified using standard methods in the art, including ammonium sulfate precipitation, affinity column chromatography, column chromatography, high-performance liquid chromatography (HPLC) purification, gel electrophoresis, etc. The antibody can be purified in the following order (see Scopes, Protein Purification (Springer-Verlag, NY, (1982)) for general purposes). The antibody in question may be substantially pure, for example, at least about 80%–85% pure, at least about 85%–90% pure, at least about 90%–95% pure, or 98%–99% or more pure, and may be free of impurities such as cell debris and macromolecules other than the antibody in question.

[0236] composition This disclosure provides compositions comprising the anti-Bb antibody of this disclosure. The antibody composition may, in addition to the antibody of this disclosure, include one or more of the following: salts, e.g., NaCl, MgCl2, KCl, MgSO4; buffers, e.g., Tris buffer, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonate sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS); solubilizers; surfactants, e.g., nonionic surfactants such as Tween-20; protease inhibitors; glycerol; etc.

[0237] nucleic acid molecules, expression vectors, and host cells This disclosure provides a nucleic acid molecule comprising a nucleotide sequence encoding the anti-Bb antibody of this disclosure.

[0238] In some embodiments, the nucleic acids of the Disclosure include a nucleotide sequence encoding a target anti-Bb antibody that includes an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:15, SEQ ID NO:23, and SEQ ID NO:30, and a light chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence.

[0239] In some embodiments, the nucleic acids of the Disclosure include a nucleotide sequence encoding a target anti-Bb antibody that includes an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:16, SEQ ID NO:24, and SEQ ID NO:31, and a heavy chain variable region that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence.

[0240] In some embodiments, the nucleic acids of the Disclosure include nucleotide sequences encoding a target anti-Bb antibody that includes a light chain variable region containing CDR-L1, CDR-L2, and CDR-L3 in one of the following combinations: SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11; SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19; and SEQ ID NO:25, SEQ ID NO:2, and SEQ ID NO:26.

[0241] In some embodiments, the nucleic acids of the Disclosure include nucleotide sequences encoding a target anti-Bb antibody that includes a heavy chain variable region containing CDR-H1, CDR-H2, and CDR-H3 in one of the following combinations: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14; SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22; SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.

[0242] In some embodiments, the nucleic acids of the Disclosure include a nucleotide sequence encoding a target anti-Bb antibody, which includes a light chain variable region and a heavy chain variable region.

[0243] A nucleic acid containing a nucleotide sequence encoding a target antibody can be operatively linked to one or more regulatory elements, such as promoters and enhancers, which enable the expression of the nucleotide sequence in the intended target cell (e.g., a cell genetically modified to synthesize the encoded antibody).

[0244] Suitable promoters and enhancer elements are known in the art. Suitable promoters for use in prokaryotic host cells include: bacteriophage T7 RNA polymerase promoter; T3 promoter; T5 promoter; lambda P promoter; trp promoter; lac operon promoter; hybrid promoters, e.g., lac / tac hybrid promoter, tac / trc hybrid promoter, trp / lac promoter, T7 / lac promoter; trc promoter; tac promoter, etc.; gpt promoter; araBAD promoter; ssaG promoter or related promoters; in vivo regulatory promoters (see, for example, U.S. Patent Publication No. 20040131637); pagC promoter (Pulkkinen and Miller, J. Bacteriol., 1991:173(1):86-93; Alpuche-Aranda et al., PNAS, 1992; 89(21):10079-83); nirB promoter (Harborne et al.) al. (1992) Mol. Micro. 6: 2805-2813), and similar works (e.g., Dunstan et al. (1999) Infect. Immun. 67: 5133-5141; McKelvie et al. (2004) Vaccine See 22:3243-3255 and Chatfield et al. (1992) Biotechnol. 10:888-892); sigma70 promoter, e.g., consensus sigma70 promoter (see, e.g., GenBank accession numbers AX798980, AX798961, and AX798183); stationary-phase promoter, e.g., dps promoter, spv promoter, etc.; promoters derived from pathogenic island SPI-2 (see, e.g., WO96 / 17951); actA promoter (see, e.g., Shetron-Rama et al. (2002) Infect. Immun. 70:1087-1096); rpsM promoter (see, e.g., Valdivia and Falkow (1996). Mol. Microbiol. 22:367); tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, for example, Melton et al. (1984) Nucl. Acids Res. 12:7035); etc., but not limited to these. Suitable potent promoters for use in prokaryotes such as Escherichia coli include Trc, Tac, T5, T7, and P ラムダ This includes, but is not limited to, those listed. Bacterial colony Non-exclusive examples of operators for use in chief cells include the lactose promoter operator (the LacI repressor protein changes its conformation when in contact with lactose, thereby preventing the LacI repressor protein from binding to the operator), the tryptophan promoter operator (when compounded with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80:21-25).

[0245] In some embodiments, for example with respect to expression in yeast cells, suitable promoters are constitutive promoters such as the ADH1 promoter, PGK1 promoter, ENO promoter, and PYK1 promoter; or modulo promoters such as the GAL1 promoter, GAL10 promoter, ADH2 promoter, PHO5 promoter, CUP1 promoter, GAL7 promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (for example, for use in Pichia).

[0246] Appropriate promoters for expression in eukaryotic cells include, but are not limited to, light chain and / or heavy chain immunoglobulin gene promoters and enhancer elements; cytomegalovirus pre-early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoters present in long terminal repeats of retrovirus origin; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art.

[0247] The selection of appropriate vectors and promoters is well within the realm of the art.

[0248] Nucleic acids containing the nucleotide sequence encoding the antibody of interest may be present in an expression vector and / or a cloning vector. This disclosure provides a recombinant vector comprising a nucleic acid containing the nucleotide sequence encoding the antibody of interest within a cloning vector. This disclosure also provides a recombinant molecule comprising a nucleic acid containing the nucleotide sequence encoding the antibody of interest, operatively ligated to an appropriate regulatory sequence(s) in the expression vector to ensure the expression of the encoded antibody. If the antibody of interest comprises two distinct polypeptides, nucleic acids containing the nucleotide sequences encoding the two polypeptides may be cloned into the same or distinct vectors to form one or more recombinant vectors. Recombinant vectors may include selectable markers, origins of replication, and other properties that provide replication and / or maintenance of the recombinant vector.

[0249] Numerous suitable vectors and promoters are known to those skilled in the art; many are commercially available for generating the recombinant molecules of interest. The following vectors are provided as examples: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0250] Expression vectors generally provide insertion of nucleic acid sequences encoding heterologous proteins, often with convenient restriction sites located near the promoter sequence. Functionally selectable markers may exist in the expression host. Suitable expression vectors include, but are not limited to, viral vectors. Examples of viral vectors include vaccinia virus; poliovirus; adenovirus (e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and See Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649; WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 00655); adeno-associated viruses (e.g., Ali et al., Hum Gene Ther 9:81 86, 1998; Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863,1997;Jomary et al.,Gene Ther 4:683 690,1997,Rolling et al.,Hum See Gene Ther 10:641 648,1999; Ali et al., Hum Mol Genet 5:591 594,1996; Srivastava, WO93 / 09239; Samulski et al., J.Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; viral vectors based on herpes simplex virus; retroviral vectors (e.g., mouse leukemia virus, splenic necrosis virus), and Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23,1997; Takahashi See et al., J Virol 73:7812 7816, 1999), retrovirus-derived vectors such as myeloproliferative sarcoma virus and mammary cancer virus; etc., but not limited to these.

[0251] As described above, the nucleic acid of interest comprises a nucleotide sequence encoding the anti-Bb antibody of this disclosure. In some embodiments, the nucleic acid of interest comprises a nucleotide sequence encoding the heavy chain and light chain CDR of the antibody of interest, wherein FR-coding nucleotide sequences are interspersed within the CDR-coding sequence. In some embodiments, the FR-coding nucleotide sequence is a human FR-coding nucleotide sequence.

[0252] host cell This disclosure provides isolated genetically modified host cells (e.g., in vitro cells) that are genetically modified with the nucleic acid of interest. This disclosure provides isolated genetically modified host cells (e.g., in vitro cells) that are genetically modified with a recombinant expression vector (may include a nucleic acid comprising a nucleotide sequence (may include) encoding the anti-Bb antibody of this disclosure). In some embodiments, the isolated genetically modified host cells of interest may produce the antibody of interest. Such cells are referred to as recombinant cells, genetically modified cells, or genetically modified host cells. The recombinant cells comprise recombinant nucleic acid (may include a nucleotide sequence (may include) (e.g., recombinant expression vector) comprising a nucleotide sequence (may include) encoding the antibody of interest.

[0253] Suitable host cells include eukaryotic host cells such as mammalian cells, insect host cells, and yeast cells; and prokaryotic cells such as bacterial cells. The introduction of target nucleic acids into host cells can be achieved, for example, by calcium phosphate precipitation, DEAE dextran-mediated transfection, liposome-mediated transfection, electroporation, or other known methods. It can be brought about by this.

[0254] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, and rodent (e.g., mouse, rat) cell lines. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) number CCL-2), Chinese hamster ovary (CHO) cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI.3), human fetal kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells, etc. In some cases, the cells are HEK cells. In some cases, the cells are CHO cells, such as CHO-K1 cells (ATCC number CCL-61), CHO-M cells, CHO-DG44 cells (ATCC number PTA-3356), etc. In some embodiments, the host cells are COS cells. In some embodiments, the host cells are 293 cells. In some embodiments, the host cells are CHO cells.

[0255] Suitable yeast cells include Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia species, Saccharomyces cerevisiae, Saccharomyces species, Hansenula polymorpha, Kluyveromyces species, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium species, Fusarium gramineum, Fusarium venenatum, Neurospora This includes, but is not limited to, *Crassa* and *Chlamydomonas reinhardtii*. In some embodiments, the host cell is *Saccharomyces*. In some embodiments, the host cell is *Pichia*.

[0256] Suitable prokaryotic cells include, but are not limited to, a variety of laboratory strains such as Escherichia coli, Bacillus (e.g., B. subtilis), and Lactobacillus species. See, for example, Carrier et al. (1992) J. Immunol. 148:1176-1181; U.S. Patent No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302. Typically, laboratory strains are non-pathogenic. In some embodiments, the host cell is Escherichia coli. In some embodiments, the host cell is Bacillus subtilis.

[0257] Pharmaceutical composition This disclosure provides compositions, including a pharmaceutical composition comprising the anti-Bb antibody of this disclosure. Generally, a pharmaceutical composition, also referred to herein as a formulation, contains an effective amount of the antibody of interest. "Effective amount" means an effective amount of the desired outcome, for example, adverse symptoms associated with complement-mediated disease or disorder. This refers to a dosage sufficient to produce a reduction, 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. In some embodiments, the antibody of interest is formulated and / or modified to allow the antibody to cross the blood-brain barrier. In some embodiments, the antibody of interest is delivered in a manner that avoids the blood-brain barrier. In some embodiments, the anti-Bb antibody of this disclosure is formulated with an active agent that promotes crossing the blood-brain barrier. In some embodiments, the antibody of interest is fused directly or via a linker to a compound that promotes crossing the blood-brain barrier.

[0258] formulation In the methods of the present disclosure, the anti-Bb antibody of the present disclosure may be administered to a host by any convenient means capable of producing the desired therapeutic or diagnostic effect. Thus, the anti-Bb antibody may be incorporated into a variety of formulations for therapeutic administration. More particularly, the antibody of interest may be formulated in a pharmaceutical composition in combination with a suitable, pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, or other pharmaceutically acceptable excipient, and may be formulated in preparations in solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols. In some embodiments, the pharmaceutical composition comprises the antibody of interest and a pharmaceutically acceptable excipient.

[0259] In pharmaceutical dosage forms, the antibodies of interest may be administered in the form of their pharmaceutically acceptable salts, or they may be used alone, or in appropriate combinations with other pharmaceutically active compounds. The following methods and excipients are illustrative and not limiting.

[0260] With regard to oral preparations, the antibody of interest may be used alone or in combination with appropriate additives, such as conventional additives like lactose, mannitol, corn starch, or potato starch; binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatin; disintegrants such as corn starch, potato starch, or sodium carboxymethylcellulose; lubricants such as talc or magnesium stearate; and, if desired, diluents, buffers, wetting agents, preservatives, and flavoring agents to prepare tablets, powders, granules, or capsules.

[0261] The antibody in question may be formulated into a preparation for injection by dissolving, suspending, or emulsifying the antibody in an aqueous or non-aqueous solvent such as vegetable oil or other similar oils, propylene glycol, synthetic fatty acid glycerides, injectable organic esters (e.g., ethyl oleate), higher fatty acids, or esters of propylene glycol; and, if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixative oils. Intravenous vehicles may include fluid and nutrient supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Furthermore, the pharmaceutical compositions of this disclosure may include additional active ingredients, such as dopamine or psychopharmacological agents, depending on the intended use of the pharmaceutical composition.

[0262] A pharmaceutical composition containing the antibody of interest is prepared by mixing the antibody of interest, having a desired degree of 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 non-toxic to the recipient at the dosage and concentration employed, and include buffers such as phosphates, citrates, and other organic acids; ascorbic acid, glutaramine, etc. Antioxidants including thion, cysteine, methionine, and citric acid; preservatives (ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propylparaben, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline, and combinations thereof; monosaccharides, disaccharides, and other carbohydrates; low molecular weight (less than approximately 10 residues) polypeptides; proteins such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants such as Tween, Brij, Pluronic, Triton-X, or polyethylene glycol (PEG).

[0263] Pharmaceutical compositions may be in liquid form, lyophilized form, or liquid form reconstituted from lyophilized form, and lyophilized preparations should be reconstituted with sterile solution before administration. The standard procedure for reconstituting a lyophilized composition is to add back a certain volume of pure water (typically equivalent to the volume removed during lyophilization); however, solutions containing antibacterial agents may be used to produce pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.

[0264] Exemplary antibody concentrations in the pharmaceutical composition in question may range from approximately 1 mg / mL to approximately 200 mg / mL, or from approximately 50 mg / mL to approximately 200 mg / mL, or from approximately 150 mg / mL to approximately 200 mg / mL.

[0265] Aqueous antibody formulations can be prepared in pH buffer solutions at pH levels ranging, for example, from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or from about 5.5. Examples of buffers suitable for pH within this range include phosphate, histidine, citrate, succinate, acetate buffers, and other organic acid buffers. Buffer concentrations can be, for example, from about 1 mM to about 100 mM or from about 5 mM to about 50 mM, depending on the buffer and the desired tonicity of the formulation.

[0266] To modulate the tonicity of the formulation, isotonic agents may be included in antibody preparations. Exemplary isotonic agents include sodium chloride, potassium chloride, glycerin, and any components from the group of amino acids and sugars, as well as combinations thereof. In some embodiments, aqueous formulations are isotonic, although hypertonic or hypotonic solutions may be appropriate. The term "isotonic" refers to a solution that has the same tonicity as some other solution to which it is compared, such as a physiological saline solution or serum. Isotonic agents may be used in amounts of about 5 mM to about 350 mM, for example, in amounts of 100 mM to 350 nM.

[0267] Surfactants may also be added to antibody formulations to reduce aggregation of the formulated antibody and / or minimize the formation of microparticles in the formulation and / or reduce adsorption. Exemplary surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Suitable examples of polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (marketed under the trademark Tween20®) and polysorbate 80 (marketed under the trademark Tween80®). Suitable examples of polyethylene-polypropylene copolymers are Pluronic® F68 or It is sold under the name Poloxamer188 (trademark). A suitable example of polyoxyethylene alkyl ether is sold under the trademark Brij (trademark). Exemplary concentrations of surfactants can range from about 0.001% to about 1% w / v.

[0268] To protect unstable active ingredients (e.g., proteins) from conditions that impair their stability during the freeze-drying process, lyoprotectants may also be added. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol, and glycerol); and amino acids (including alanine, glycine, and glutamic acid). Lyoprotectants may be included in amounts of about 10 mM to 500 nM.

[0269] In some embodiments, the formulation in question comprises the antibody in question and one or more of the active ingredients identified above (e.g., surfactants, buffers, stabilizers, isotonic agents), and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propylparaben, benzalkonium chloride, and combinations thereof. In other embodiments, preservatives are included in the formulation at concentrations ranging, for example, from about 0.001% to about 2% (w / v).

[0270] For example, the formulation in question may be a liquid or lyophilized formulation suitable for parenteral administration, and may contain approximately 1 mg / mL to approximately 200 mg / mL of the antibody in question; approximately 0.001% to approximately 1% of at least one surfactant; approximately 1 mM to approximately 100 mM of buffer; optionally, approximately 10 mM to approximately 500 mM of stabilizer; and approximately 5 mM to approximately 305 mM of isotonic agent, and have a pH of approximately 4.0 to approximately 7.0.

[0271] As another example, the parenteral formulation in question is a liquid or lyophilized formulation containing approximately 1 mg / mL to approximately 200 mg / mL of the target antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose, and has a pH of 5.5.

[0272] As another example, the parenteral formulation in question may contain: 1) 15 mg / mL of the target antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose, with a pH of 5.5; or 2) 75 mg / mL of the target antibody; 0.04% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose, with a pH of 5.5; or 3) 75 mg / mL of the target antibody; 0.02% Tween 20 w / v; 20 mM L-histidine; and 250 mM sucrose, with a pH of 5.5; or 4) 75 mg / mL of the target antibody; 0.04% Tween 20 w / v; 20 mM The preparation includes a lyophilized formulation containing L-histidine and 250 mM trehalose, with a pH of 5.5; or 75 mg / mL of the target antibody; 0.02% Tween 20 w / v; 20 mM L-histidine and 250 mM trehalose, with a pH of 5.5.

[0273] As another example, the parenteral formulation in question may contain: 1) 7.5 mg / mL of the target antibody; 0.02% Tween20 w / v; 120 mM L-histidine; and 250 mM sucrose, with a pH of 5.5; or 2) 37.5 mg / mL of the target antibody; 0.02% Tween20 w / v; 10 mM L-histidine; and 125 mM sucrose, with a pH of 5.5; or 3) 37.5 mg / mL of the target antibody; 0.01% Tween20 w / v; 10 mM L-histidine; and 125 mM sucrose, with a pH of 5.5; or 4) 37.5 mg / mL of the target antibody; 0.02% Tween20 w / v; 10 mM L-histidine; containing 125 mM trehalose and having a pH of 5.5; or 5) 37.5 mg / mL of the target antibody; 0.01% Tween 20 w / v; containing 10 mM L-histidine and 125 mM trehalose and having a pH of 5.5 7) A solution containing 75 mg / mL of the target antibody, 0.02% Tween 20 w / v, 20 mM L-histidine, and 250 mM trehalose, with a pH of 5.5; or 8) A solution containing 75 mg / mL of the target antibody, 0.02% Tween 20 w / v, 20 mM L-histidine, and 250 mM mannitol, with a pH of 5.5; or 9) A solution containing 75 mg / mL of the target antibody, 0.02% Tween 20 w / v, 20 mM L-histidine, and 140 mM sodium chloride, with a pH of 5.5; or 150 mg / mL of the target antibody, 0.02% Tween 20 w / v, 20 mM A liquid formulation containing L-histidine and 250 mM trehalose, with a pH of 5.5; or 10) 150 mg / mL of the target antibody; 0.02% Tween20 w / v; 20 mM L-histidine and 250 mM mannitol, with a pH of 5.5; or 11) 150 mg / mL of the target antibody; 0.02% Tween20 w / v; 20 mM L-histidine and 140 mM sodium chloride, with a pH of 5.5; or 12) 10 mg / mL of the target antibody; 0.01% Tween20 w / v; 20 mM L-histidine and 40 mM sodium chloride, with a pH of 5.5.

[0274] The antibody in question may be used in aerosol formulations administered by inhalation. The antibody may be formulated in acceptable pressurized propellants such as dichlorodifluoromethane, propane, or nitrogen. Aerosol formulations, such as nasal spray formulations, contain a purified aqueous solution or other solution of the active ingredient, along with preservatives and isotonic agents. Such formulations are adjusted to a pH and isotonic state compatible with the nasal mucosa.

[0275] Furthermore, the target antibody can be prepared in a suppository by mixing it with a variety of bases, such as emulsifying or water-soluble bases. The target antibody can be administered rectally via a suppository. The suppository may contain a vehicle such as cocoa butter, carbowax, and polyethylene glycol, which melts at body temperature but solidifies at room temperature.

[0276] Unit dosage forms for oral or rectal administration may be provided, such as syrups, elixirs, and suspensions, with each dosing unit, e.g., a teaspoon, a tablespoon, a tablet, or a suppository, containing a predetermined amount of the composition. Similarly, unit dosage forms for injection or intravenous administration may contain the antibody of interest in the composition as a solution in sterile water, ordinary saline, or another pharmaceutically acceptable carrier.

[0277] As used herein, the term “unit dosage form” refers to physically separate units suitable as unit doses for human and animal subjects, each unit containing a predetermined amount of the calculated anti-Bb antibody of this disclosure in an amount sufficient to produce the desired effect when used with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the antibody of the subject may depend on the specific antibody employed, the effect to be achieved, and the pharmacodynamics associated with each antibody in the host.

[0278] Other methods of administration may also find use in conjunction with the methods of this disclosure. For example, the antibody of interest may be formulated in suppositories and, in some cases, in aerosols and intranasal compositions. With respect to suppositories, the vehicle composition may include traditional binders and carriers, such as polyalkylene glycols or triglycerides. Such suppositories may be formed from a mixture containing the active ingredient in a range of about 0.5% to about 10% (w / w), for example, about 1% to about 2%.

[0279] Intranasal preparations typically contain a vehicle that does not irritate the nasal mucosa and does not significantly interfere with ciliary function. Diluents such as water, aqueous saline, or other known substances may be used. Nasal preparations may also contain preservatives, such as but not limited to chlorobutanol and benzalkonium chloride. Surfactants may be present to enhance the absorption of the target antibody by the nasal mucosa.

[0280] The target antibody may be administered as an injectable formulation. Typically, the injectable composition is prepared as a solution or suspension; a solid form suitable for the solution or suspension in a liquid vehicle before injection may also be prepared. The preparation may also be emulsified, or the antibody may be encapsulated in a liposome vehicle.

[0281] Suitable excipient vehicles include, for example, water, saline, dextrose, glycerol, ethanol, and combinations thereof. In addition, if desired, the vehicle may contain small amounts of auxiliary substances such as wetting agents, emulsifiers, or pH buffers. Practical methods for preparing such dosage forms are known or obvious to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985. The composition or formulation to be administered will, in any case, contain a sufficient amount of the antibody of the subject to achieve the desired state in the subject being treated.

[0282] Pharmacovigilantly acceptable excipients such as vehicles, adjuvants, carriers, or diluents are readily available for public use. Furthermore, pharmaceutically acceptable auxiliary substances such as pH adjusters and buffers, tonicity adjusters, stabilizers, and wetting agents are readily available for public use.

[0283] In some cases, the antibody of interest may be formulated in a controlled-release formulation. Sustained-release preparations may be prepared using methods well known in the art. Suitable examples of sustained-release preparations include semipermeable matrices of hydrophobic solid polymers containing antibodies, in which the matrix is ​​in the form of a molded article, e.g., a film or microcapsules. Examples of sustained-release matrices include polyesters, L-glutamic acid and ethyl-L-glutamate copolymers, non-degradable ethylene vinyl acetate, hydrogels, polylactides, degradable lactate-glycolic acid copolymers, and poly-D-(-)-3-hydroxybutyrate. Possible loss of biological activity and possible changes in immunogenicity of antibodies contained in sustained-release preparations can be prevented by using appropriate additives, by controlling the water content, and by developing specific polymer matrix compositions.

[0284] Within the scope of this disclosure, controlled release may be understood to mean any one of numerous long-release dosage forms. The following terms may be considered substantially equivalent to controlled release for the purposes of this disclosure: continuous release, controlled release, delayed release, depot, long-release, stepwise release, immediate release, prolonged release, programmed release, sustained release, proportional release, long-release, repository, delayed, gradual release, intervald release, sustained release, time coat, timed release, delayed action, long-acting action, stratified time action, long-acting action, sustained action, repeated action, gradual action, sustained action, and sustained-acting pharmaceuticals. Further discussion of these terms can be found in Lesczek Krowczynski, Extended-Release Dosage Forms, 1987 (CRC Press, Inc.).

[0285] Various controlled-release technologies cover a very wide range of drug dosage forms. Controlled-release technologies include, but are not limited to, physical and chemical systems.

[0286] Physical systems include storage systems with rate-controlled membranes, such as microencapsulation, macroencapsulation, and membrane systems; storage systems without rate-controlled membranes, such as hollow fibers, ultramicroporous cellulose triacetate, and porous polymeric substrates and foams; and such systems physically dissolved in nonporous, polymeric, or elastic matrices (e.g., non-erosive, erosive, environmental agent ingression, and degradable), and nonporous, polymeric, or elastic matrices. Integrated systems including materials physically dispersed in a casing (e.g., non-erosive, erosive, environmentally friendly, and degradable); layered structures including a reservoir that is chemically similar or dissimilar to an outer control layer; and other physical methods such as osmotic pumps or adsorption to ion exchange resins.

[0287] Chemical systems include, but are not limited to, chemical erosion of the polymer matrix (e.g., heterogeneous or homogeneous erosion) or biological erosion of the polymer matrix (e.g., heterogeneous or homogeneous). Further considerations regarding the classification of systems for controlled release can be found in Agis F. Kydonieus, Controlled Release Technologies: Methods, Theory and Applications, 1980 (CRC Press, Inc.).

[0288] Several controlled-release drug formulations have been developed for oral administration. These include, but are not limited to, membrane-permeable gastrointestinal delivery systems, including osmotically controlled gastrointestinal delivery systems; hydrodynamically controlled gastrointestinal delivery systems; microporous membrane-permeable gastrointestinal delivery systems; gastric juice-resistant intestinal target controlled-release gastrointestinal delivery systems; gel diffusion-controlled gastrointestinal delivery systems; and ion-exchange-controlled gastrointestinal delivery systems, including cationic and anionic drugs. Additional information on controlled-release drug delivery systems can be found in Yie W. Chien, Novel Drug Delivery Systems, 1992 (Marcel Dekker, Inc.).

[0289] Dosage The appropriate dosage can be determined by the attending physician or other qualified healthcare professional based on various clinical factors. As is well known in the field of medicine, the dosage for any given patient depends on many factors, including the patient's size, body surface area, age, the specific compound being administered, the patient's sex, the time and route of administration, overall health status, and other medications being administered concurrently. The target anti-Bb antibody may be administered in amounts ranging from 1 ng / kg body weight to 20 mg / kg body weight per dose, for example, 0.1 mg / kg body weight to 10 mg / kg body weight, for example, 0.5 mg / kg body weight to 5 mg / kg body weight; however, doses below or above this exemplary range may be considered, taking into account the aforementioned factors in particular. In some cases, the anti-Bb antibody of this disclosure is administered in doses of 20 mg / kg body weight to 100 mg / kg; for example, in some cases, the anti-Bb antibody of this disclosure is administered in doses of 20 mg / kg to 25 mg / kg, 25 mg / kg to 30 mg / kg, 30 mg / kg to 35 mg / kg, 35 mg / kg to 40 mg / kg, 40 mg / kg to 45 mg / kg, 45 mg / kg to 50 mg / kg It is administered in amounts of 50 mg / kg to 55 mg / kg, 55 mg / kg to 60 mg / kg, 60 mg / kg to 65 mg / kg, 65 mg / kg to 70 mg / kg, 70 mg / kg to 75 mg / kg, 75 mg / kg to 80 mg / kg, 80 mg / kg to 85 mg / kg, 85 mg / kg to 90 mg / kg, 90 mg / kg to 95 mg / kg, or 95 mg / kg to 100 mg / kg body weight. In some cases, the anti-Bb antibody of this disclosure is administered in amounts of 20 mg / kg to 40 mg / kg body weight per dose. In some cases, the anti-Bb antibody of this disclosure is administered in amounts of 40 mg / kg to 60 mg / kg body weight per dose. If the regimen is a continuous infusion, it may also be in the range of 1 μg to 10 mg per kilogram of body weight per minute. In some cases, the anti-Bb antibody of this disclosure is administered in a weight-independent amount.In some cases, the anti-Bb antibody of this disclosure is administered in amounts of 50 mg to 500 mg per dose or per total daily dose; for example, 50 mg to 75 mg, 75 mg to 100 mg, 100 mg to 150 mg, 150 mg to 200 mg, 200 mg to 250 mg, 250 mg to 300 mg, 300 mg to 400 mg, or 400 mg to 500 mg per dose or per total daily dose.

[0290] In some cases, the dose of the anti-Bb antibody of this disclosure is 0.001 μg to 1000 μg. While within the specified range, doses below or above this exemplary range are conceivable, particularly considering the aforementioned factors. In some cases, the dosage may range from, for example, about 0.0001 to 100 mg / kg, or about 0.01 to 5 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, etc.) per body weight. For example, the dosage may be 1 mg / kg body weight or 10 mg / kg body weight, or within the range of 1 to 10 mg / kg, or at least 1 mg / kg. Doses that fall within the above ranges are also intended to be within the scope of the present invention.

[0291] Individuals may be administered such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. Exemplary treatments involve multiple doses over a long period, for example, at least six months. Additional exemplary treatment regimens involve administration every two weeks, once a month, or once every three to six months. Exemplary dosing schedules include 1–10 mg / kg or 15 mg / kg on consecutive days, 30 mg / kg every other day, or 60 mg / kg weekly. In some methods, two or more types of monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered falls within the indicated range. Progress can be monitored by periodic assessments.

[0292] Those skilled in the art will readily understand that dose levels and administration schedules can vary as a function of specific antibodies, symptom severity, and the subject's susceptibility to side effects. Preferred dosages and administration schedules for a given compound can be readily determined by those skilled in the art through various means.

[0293] Route of administration The target antibody is administered to the individual using any available method and route appropriate for drug delivery, including in vivo and ex vivo methods, as well as systemic and local routes of administration.

[0294] Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subarachnoid, 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 routes of administration. The routes of administration may be combined and, if desired, modified depending on the antibody and / or desired effect. The antibody composition of interest may be administered in single or multiple doses. In some cases, the antibody composition of interest is administered orally. In some cases, the antibody composition of interest is administered via inhalation. In some cases, the antibody composition of interest is administered intranasally. In some cases, the antibody composition of interest is administered topically. In some cases, the antibody composition of interest is administered intracranially. In some cases, the antibody composition of interest is administered intravenously. In some cases, the antibody composition of interest is administered into the subarachnoid space. In some cases, the antibody composition of interest is administered subcutaneously. In some cases, the antibody composition of interest is administered intramuscularly.

[0295] The antibodies of this disclosure may be administered to a host by any available conventional method and route suitable for conventional drug delivery, including systemic or local routes. Generally, the routes of administration intended by the present invention include, but are not limited to, enteral, parenteral, or inhalation routes.

[0296] Parenteral routes of administration other than inhalation include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intrathecal, intrasternal, subarachnoid, and intravenous routes, i.e., any route of administration other than through the gastrointestinal tract. Parenteral administration may be performed to result in systemic or local delivery of the target antibody. When systemic delivery is desired, administration is typically invasive or systemically absorbed topical or mucosal administration of the pharmaceutical preparation. It is accompanied by.

[0297] The target antibody can also be delivered to the target population by intraintestinal administration. Intestinal routes of administration include, but are not limited to, oral and rectal (e.g., using suppositories) delivery.

[0298] Treatment means at least improvement of the symptoms associated with the pathological condition that afflicts the host, and improvement is used in a broad sense to mean at least a reduction in the magnitude of parameters associated with the pathological condition being treated, such as symptoms, including complement-mediated diseases or disorders. As such, treatment also includes situations in which the pathological condition or at least the symptoms associated with it are completely inhibited, for example, prevented from occurring or stopped, or terminated, so that the host is no longer afflicted with the pathological condition or at least the symptoms that characterize the pathological condition.

[0299] In some cases, the antibody in question is administered by injection and / or delivery, for example, directly into a site within a cerebral artery or into brain tissue. The antibody in question may also be administered directly to the target site, for example, by biolistic delivery to the target site.

[0300] A diverse range of hosts (the term “host” is used herein interchangeably with the terms “subject,” “individual,” and “patient”) can be treated according to the methods of the subject. Generally, such hosts are “mammals” or “mammals,” terms used broadly to describe organisms within the class Mammalia, including the orders Carnivores (e.g., cats), Herbivores (e.g., cattle, horses, and sheep), Omnivores (dogs, goats, and pigs), Rodents (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In some cases, the host is an individual with a complement system, such as a mammal, fish, or invertebrate. In some cases, the host 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 cases, the host is a human.

[0301] This disclosure provides a suitable container for containing a composition comprising a target anti-Bb antibody for administration to an individual. For example, the target antibody may be placed in a suitable container for containing the pharmaceutical composition. The container may be, for example, a bottle (e.g., having a sealing device such as a lid), a blister pack (e.g., it may provide single or multiple doses per blister), a vial, flexible packaging (e.g., a sealed Mylar bag or plastic bag), an ampoule (for a single dose in solution), a dropper, a syringe, a thin film, a tube, etc. In some cases, the container, such as a sterile container, contains the target pharmaceutical composition. In some cases, the container is a bottle or a syringe. In some cases, the container is a bottle. In some cases, the container is a syringe.

[0302] For example, kits containing a unit dose of the target antibody are provided, for oral or injectable medication. Such kits include, in addition to a container containing the unit dose, an informational leaflet describing the use of the antibody in treating the pathological condition of interest and the associated benefits. Preferred compounds and unit doses are those described above herein.

[0303] Methods for treating complement-mediated disorders or conditions This disclosure provides a method for treating complement-mediated disorders. The method generally involves administering an effective dose of the anti-Bb antibody of this disclosure to an individual in need of administration. In some cases, administration of the anti-Bb antibody modulates AP activity in the individual's cells, tissues, or body fluids, thereby treating the complement-mediated disorder.

[0304] In some cases, the effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting 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% the AP activity in cells, tissues, or body fluids of an individual, compared to the level of AP activity in cells, tissues, or body fluids in the absence of or before the administration of the anti-Bb antibody. In some cases, the anti-Bb antibody 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 activity.

[0305] In some cases, an effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting 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% the formation of MAC in the cells, tissues, or body fluids of an individual, compared to the amount of MAC formed in the cells, tissues, or body fluids in the absence of or before the administration of the anti-Bb antibody.

[0306] In some cases, an effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting 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% of C3b / Bb-mediated cleavage of C3 in cells, tissues, or body fluids of an individual, compared to C3 cleavage in cells, tissues, or body fluids in the absence of or before administration of the anti-Bb antibody. In some cases, the anti-Bb 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 C3b / Bb-mediated cleavage of C3.

[0307] In some cases, an effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting C3b / Bb-mediated cleavage of C3, thereby reducing the production of C3 cleavage products. For example, in some cases, an effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting C3b / Bb-mediated cleavage of C3, thereby reducing the production of C3 cleavage products (e.g., C3a and / or C3b) in the cells, tissues, or body fluids of an individual by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% compared to the production of C3 cleavage products in cells, tissues, or body fluids in the absence of or before the administration of the anti-Bb antibody.

[0308] In some cases, an effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting complement AP-mediated lysis of cells in an individual by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, compared to the degree of cell lysis in the absence of or before administration of the anti-Bb antibody. In some cases, the anti-Bb 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 dissolution.

[0309] In some cases, an effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting complement AP-mediated hemolysis in the cells, tissues, or body fluids (e.g., RBC-containing body fluids) of an individual by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, compared to the degree of hemolysis in the cells, tissues, or body fluids in the absence of or before the administration of the anti-Bb antibody. In some cases, the anti-Bb 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 hemolysis.

[0310] In some cases, the effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting the production of anaphylatoxin. For example, in some cases, the effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting the production of C3a and C5a 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 more than 95%, compared to the amount of C3a and C5a produced in the absence of or before the administration of the anti-Bb antibody.

[0311] In some cases, an effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting 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% the AP-mediated deposition of C3b, C3d, or other C3-resolution products onto cells or tissues in an individual, compared to the amount of deposition of C3b, C3d, or other C3-resolution products onto cells or tissues in the absence of or before administration of the anti-Bb antibody. In some cases, the anti-Bb 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 deposition of C3b, C3d, or other C3 resolution products onto cells or tissues.

[0312] In some cases, an effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting AP-mediated C3b deposition on cells or tissues in an individual by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, compared to the amount of C3b deposition on cells or tissues in the absence of or before administration of the anti-Bb antibody. In some cases, the anti-Bb 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 -8M, 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.

[0313] In some cases, the effective amount of the anti-Bb antibody of this disclosure is 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%, and at least 85% of the AP-mediated deposition of C3b, C3d, or other C3 fragmentation products on RBCs in an individual, compared to the amount of deposition of C3b, C3d, or other C3 fragmentation products on RBCs in an individual in the absence of or before administration of the anti-Bb antibody. The amount is effective in inhibiting %, at least 90%, at least 95%, or 100%. In some cases, the anti-Bb 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 deposition of C3b, C3d, or other C3 fragmentation products onto RBCs.

[0314] In some cases, an effective dose of the anti-Bb antibody of this disclosure is an amount effective in inhibiting AP-mediated C3b deposition on RBCs in an individual by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%, compared to the amount of C3b deposition on RBCs in the individual in the absence of or before administration of the anti-Bb antibody. In some cases, the anti-Bb 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.

[0315] In some cases, the anti-Bb antibodies of the Disclosure, when administered in one or more doses to an individual in need of treatment, reduce the amount of circulating Bb factor in that individual. For example, in some cases, the anti-Bb antibodies of the Disclosure, when administered in one or more doses to an individual in need of treatment, reduce the amount of circulating Bb factor in that individual by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the amount of circulating Bb factor in that individual in the absence of the anti-Bb antibody administration, or compared to the amount of circulating Bb factor in that individual before the administration of the anti-Bb antibody.

[0316] In some cases, the anti-Bb antibodies of the Disclosure, when administered in one or more doses to an individual in need of treatment, reduce the amount of Bb factor in the plasma of that individual. For example, in some cases, the anti-Bb antibodies of the Disclosure, when administered in one or more doses to an individual in need of treatment, reduce the amount of Bb factor in the plasma of that individual by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% compared to the amount of Bb factor in the plasma of that individual in the absence of the anti-Bb antibody administration, or compared to the amount of Bb factor in the plasma of that individual before the administration of the anti-Bb antibody.

[0317] In some cases, the methods of the present disclosure for treating an individual having a complement-mediated disease or disorder include administering to the individual an anti-Bb antibody of the present disclosure, or a pharmaceutical composition comprising a) the anti-Bb antibody of the present disclosure; and b) a pharmaceutically acceptable excipient suitable for administration to such individual. In some cases, the individual is a mammal. In some cases, the individual is a human. Administration may be by any route known to those skilled in the art, including those disclosed herein. In some cases, administration is intravenous. In some cases, administration is subarachnoid. In some cases, administration is intramuscular. In some cases, administration is subcutaneous. In some cases, the antibody is humanized.

[0318] Complement-mediated diseases and disorders that are appropriate for treatment with the anti-Bb antibodies of this disclosure include diseases and disorders associated with alternative complement pathways.

[0319] Complement-mediated diseases and disorders that are appropriate for treatment with the anti-Bb antibodies of this disclosure include, but are not limited to, paroxysmal nocturnal hemoglobinuria (PNH), idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), disseminated intravascular coagulation (DIC), antiphospholipid syndrome (APS), post-transfusion purpura, neonatal alloimmune thrombocytopenia (NAITP), ischemia / reperfusion injury, and age-related macular degeneration (AMD).

[0320] Complement-mediated diseases and disorders that are appropriate for treatment with the anti-Bb antibodies of this disclosure include, but are not limited to, asthma, atypical hemolytic uremic syndrome (aHUS), fulminant antiphospholipid syndrome (Asherson's syndrome; or CAPS), densitosis (DDD), C3 glomerulonephritis (C3GN), age-related macular degeneration (AMD), dry AMD, wet AMD, acquired epidermolysis bullosa, rheumatoid arthritis, membranoproliferative glomerulonephritis type II, and paroxysmal nocturnal hemoglobinuria (PNH).

[0321] In some cases, complement-mediated disorders are selected from the group consisting of ischemia-reperfusion injury, atypical hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, paroxysmal nocturnal hemoglobinuria, densitosis, age-related macular degeneration, spontaneous abortion, pauci-immune vasculitis, epidermolysis bullosa, recurrent abortion, multiple sclerosis, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis, Degos disease, Graves' disease, Hashimoto's thyroiditis, type 1 diabetes mellitus, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome, multifocal motor neuropathy, neuromyelitis optica, antiphospholipid syndrome, and fulminant antiphospholipid syndrome.

[0322] AP-related disorders and classical pathway (CP)-related disorders include, for example, ischemia-reperfusion injury, atypical hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, paroxysmal nocturnal hemoglobinuria, densitosis, age-related macular degeneration, spontaneous abortion, oligoimmune vasculitis, epidermolysis bullosa, recurrent abortion, multiple sclerosis, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis, Degos disease, Graves' disease, Hashimoto's thyroiditis, type 1 diabetes mellitus, psoriasis, pemphigus, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, Goodpasture syndrome, multifocal motor neuropathy, neuromyelitis optica, antiphospholipid syndrome, and fulminant antiphospholipid syndrome.

[0323] Complement-related disorders include, but are not limited to, asthma, bronchitis, chronic obstructive pulmonary disease (COPD), interstitial lung disease, alpha-1 antitrypsin deficiency, emphysema, bronchiectasis, bronchiolitis obstructive, alveolitis, sarcoidosis, pulmonary fibrosis, and collagen vascular disorders, as well as other complement-related lung disorders.

[0324] Complement-mediated diseases and disorders suitable for treatment with the anti-Bb antibody of this disclosure include age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, argyrophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behçet's disease, British amyloid angiopathy, bullous pemphigus, Buerger's disease, C1q nephropathy, cancer, fulminant antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, and Creutzfeldt-Kuythorne syndrome. Tojakob disease, Crohn's disease, cryoglobulinemia-associated vasculitis, Boxer dementia, dementia with Lewy bodies (DLB), diffuse neurofibrillary tangles with calcification, discoid lupus erythematosus, Down syndrome, focal segmental glomerulosclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia with parkinsonism linked to chromosome 17, frontotemporal lobar degeneration, Gerstmann-Streussler-Scheinker disease, Guillain-Barré syndrome, Hallervondel-Spatz disease, hemolytic uremic syndrome, hereditary angioedema, hypophosphatemia, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infection Sexually transmitted diseases (e.g., diseases caused by bacteria (e.g., Neisseria meningitidis or Streptococcus), viruses (e.g., human immunodeficiency virus (HIV)), or other infectious agents), inflammatory diseases, ischemia / reperfusion injury, mild cognitive impairment, immune thrombocytopenic purpura (ITP), molybdenum cofactor deficiency (MoCD) type A, membranoproliferative glomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis (MPGN) II (dense glomerulonephritis), membranous glomerulonephritis, polyinfarct dementia, lupus (e.g., systemic lupus erythematosus (SLE)), glomerulonephritis, Kawasaki disease, multifocal motor neuropathy, polyinfarct Myasthenia gravis, multiple system atrophy, myasthenia gravis, myocardial infarction, myotonic dystrophy, neuromyelitis optica, Niemann-Pick disease type C, non-Guam type motor neuron disease with neurofibrillary tangles, Parkinson's disease, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, post-encephalitis parkinsonism, polymyositis, prion protein brain amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga toxin Escherichia coli (STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, dementia with only tangles (Tangle This includes, but is not limited to, dementia (only dementia), transplant rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegener's granulomatosis, sickle cell disease, cryoglobulinemia, mixed cryoglobulinemia, essential mixed cryoglobulinemia, type II mixed cryoglobulinemia, type III mixed cryoglobulinemia, nephritis, lupus nephritis, bullous pemphigoid, acquired epidermolysis bullosa, delayed hemolytic transfusion reactions, and platelet refractory disease.

[0325] Combination therapy The anti-Bb antibodies described herein may be administered to individuals in need of administration, either alone (e.g., as monotherapy) or in combination with one or more additional therapeutic agents.

[0326] As used herein, “in combination with” refers to, for example, cases where the first compound is administered during the entire course of administration of the second compound; cases where the first compound is administered during a period that overlaps with the administration of the second compound, for example, where the administration of the first compound begins before the administration of the second compound and ends before the administration of the second compound is completed; cases where the administration of the second compound begins before the administration of the first compound and ends before the administration of the first compound is completed; cases where the administration of the first compound begins before the administration of the second compound begins and ends before the administration of the first compound is completed; and cases where the administration of the second compound begins before the administration of the first compound begins and ends before the administration of the second compound is completed. As such, “in combination with” may also refer to regimens involving the administration of two or more types of compounds. As used herein, “in combination with” also refers to the administration of two or more types of compounds that can be administered in the same or different formulations, by the same or different routes, and in the same or different dosage forms.

[0327] Individuals to be treated Individuals suitable for treatment with the target anti-Bb antibody include individuals diagnosed with complement-mediated disease or disorder; individuals at higher risk than the general population of developing complement-mediated disease or disorder (e.g., individuals with a genetic predisposition to develop complement-mediated disease or disorder); individuals with Parkinson's disease with dementia (PDD); individuals with Alzheimer's disease; individuals with paroxysmal nocturnal hemoglobinuria (PNH); individuals with idiopathic thrombocytopenic purpura (ITP); individuals with thrombotic thrombocytopenic purpura (TTP); individuals with hemolytic uremic syndrome (HUS); individuals with disseminated intravascular coagulation (DIC); individuals with antiphospholipid syndrome (APS); individuals with post-transfusion purpura; individuals with neonatal alloimmune thrombocytopenia (NAITP); individuals with ischemia / reperfusion injury; and individuals with age-related macular degeneration (AMD). This also includes individuals who have one or more of the disabilities.

[0328] In some cases, the individual is an adult. In some cases, an adult is 20 years of age or older, 30 years of age or older, 40 years of age or older, 50 years of age or older, 60 years of age or older, 70 years of age or older, or 80 years of age or older. For example, an adult may be 20-30 years of age, 30-40 years of age, 40-50 years of age, 50-60 years of age, 60-70 years of age, or may be over 70 years of age. In some cases, the individual is a human child. In some cases, a human child is under 20 years of age, under 10 years of age, or under 5 years of age. [Examples]

[0329] The following examples are provided to give a complete disclosure and explanation of how the present invention is carried out and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to represent that the experiments below are all or only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantity, temperature, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are by weight, molecular weight is weight-average molecular weight, temperature is at Celsius, and pressure is in or near air. Standard abbreviations may be used. For example, bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; im, intramuscular; ip, intraperitoneal; sc, subcutaneous; etc.

[0330] Example 1: Production of anti-Bb factor monoclonal antibody Anti-Bb factor monoclonal antibodies ("Bb factor mAbs") M4, M10, M12, M17, M18, and M20 were produced as follows: A hybridoma library was generated by immunization of NZBW mice with purified human Bb factor protein and screened for binding to targets using techniques known to those skilled in the art (antibody solution; see, e.g., Galfre et al., Methods in Enzymology 73:346 (1981)). Single-cell clones were generated using flow cytometry, and the supernatants from these individual clones were screened for preferential binding of Bb factor to full-length factor B by direct enzyme-linked immunosorbent assay (ELISA), such as the one disclosed in, e.g., Nix et al., Immunoassays, A Practical Approach, editor JPGosling, pp.239-261, Oxford University Press (2000). Twenty clones were selected that showed varying degrees of specific binding to factor Bb compared to factor B. These clones were expanded in culture, and monoclonal antibodies were purified from the hybridoma supernatant. The purified mAbs were further screened for inhibition of alternative pathway (AP) activity using the Complement System Alternative Pathway WIESLAB kit (Euro Diagnostica, Sweden). Based on these results, six clones were selected for further analysis.

[0331] Example 2: Binding of anti-Bb factor mAb to human Bb factor. Relative EC to factor Bb mAb 50 The values ​​were determined by ELISA. Unlabeled purified human Bb factor (Complement Technologies; 1 μg / mL) was coated onto a high-binding ELISA plate and incubated with incremental amounts of purified mAb (3-fold serial dilutions starting at 10 μg / mL). Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Southern Biotech) was tested. The reaction was carried out using 3,3',5,5'-tetramethylbenzidine (TMB) 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Scientific). The reaction was stopped with an equal volume of 1N sulfuric acid; OD 450nm The absorbance was measured at each monoclonal. The data is shown in Figure 1. EC 50 This was calculated using PRISM software; EC 50 The values ​​are shown in Figure 2. All mAbs showed excellent affinity, and most were in the sub-nanomolar range of EC. 50 It had.

[0332] Figure 1. Binding of purified anti-Bb factor mAb to human Bb factor.

[0333] Figure 2. Binding affinity of anti-Bb factor mAbs to human Bb factor.

[0334] Example 3: Specificity of anti-Bb factor mAb against activated Bb factor The preferential binding of a Bb factor antibody to activated factor B versus full-length factor B enzyme precursor was determined using an optimized antigen capture ELISA format to evaluate binding to the target soluble protein. A 1 μg / mL antibody was conjugated to a goat anti-mouse IgG coated plate (Pierce; Thermo Scientific), followed by incubation with 1 μg / mL biotinylated Bb factor or B factor (Complement Technologies). The bound Bb / B factor proteins were detected with streptavidin-HRP and reacted with TMB 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Scientific). The reaction was stopped with an equal volume of 1N sulfuric acid; OD 450nmAbsorbance was measured. The ratio of absorbance due to binding of factor Bb to factor B (fBb / B ratio) was calculated, and the results are shown in Figure 3. M17 showed virtually perfect specificity for factor Bb and did not have detectable binding to factor B in this assay. M10 and M18 showed similar preferences in this assay (fBb / B ratios of 5.5 and 8.4, respectively), while M4, M12, and M20 showed no preference (fBb / B ratio ≥ 1).

[0335] The binding of Bb factor mAbs to full-length B factor in solution was evaluated by size exclusion chromatography (SEC). For SEC, 100 μg / mL of purified B factor was incubated with purified mAbs in up to 3-fold Mohler excess. After incubation at room temperature (RT) for 1 hour, the samples were loaded onto a TSKgel G3000SWxl gel filtration column. The presence of new, higher molecular weight peaks in the chromatogram, as well as a decrease in the free B factor and free antibody peaks, indicated antigen:antibody complex formation. The SEC results correlated well with the binding specificity determined by antigen capture ELISA (Figure 4). M10, M17, and M18, which showed specificity for Bb factor by ELISA, did not bind to full-length B factor in SEC; M4, M12, and M20, which did not show binding specificity by ELISA, all similarly bound to full-length B factor in SEC.

[0336] Figure 3. Binding of purified anti-Bb factor mAb to soluble factor B and factor Bb.

[0337] Figure 4. Specificity of binding of anti-Bb factor mAbs to B factor versus B factor as determined by ELISA, compared with results obtained by size exclusion chromatography (SEC) (Figure 3).

[0338] Example 4: Binding of anti-Bb factor mAb to cynomolgus monkey protein Bb factor mAbs were assayed by ELISA for their ability to bind to cynomolgus monkey B factor or Bb factor. Each purified protein (Innovative Research; in-house purified) at 5 μg / mL was coated onto an ELISA plate and incubated with purified Bb factor mAbs (3-fold serial dilutions starting at 10 μg / mL) to determine binding. The antibodies present were detected in the same manner as in Example 2. EC against factor Bb versus factor B 50 This was calculated using Prism software and is shown in Figure 5.

[0339] Figure 5. Binding of purified anti-Bb factor mAb to purified cynomolgus monkey B factor or Bb factor.

[0340] Example 5: Inhibition of the complement alternative pathway (AP) by anti-Bb factor mAb Inhibition of complement AP activity by factor Bb mAbs was measured using the Complement System Alternative Pathway WIESLAB® kit. This is a plate-based assay that specifically activates the alternative pathway using lipopolysaccharide (LPS), with readout being the deposition of the final membrane invasion complex (MAC) over time. 11% normal human serum (Complement Technologies) was incubated with 2-fold serial dilutions of mAbs or mouse isotype controls starting at 200 μg / mL. max The results were determined for each concentration and are shown in Figure 6. All anti-Bb factor monoclonal antibodies were able to inhibit AP-mediated MAC deposition, with M4 / M12 > M20 > M10 / M18 > M17.

[0341] The inhibition of AP pathway-mediated hemolysis and C3b deposition by factor Bb mAbs was determined using human serum and rabbit erythrocytes in a buffer containing EGTA, which inhibits the classical pathway. Anti-factor Bb mAbs or mouse isotype controls (2-fold dilutions starting at 100 μg / mL) were incubated with 10% human serum and rabbit erythrocytes (RBCs) at 37°C for 1 hour. The supernatant A 540nmThe amount of lysis was determined by measuring the absorbance and subtracting the background absorbance in a control well containing ethylenediaminetetraacetic acid (EDTA). The results are shown in Figure 7. All Bb factor mAbs were able to inhibit AP-mediated hemolysis, with M4 / M12 > M20 > M10 / M18 > M17.

[0342] To assay C3 resolution product deposition on red blood cells (RBCs), cell pellets were stained with an anti-C3b antibody (6C9; Thermo Scientific), positive cells were detected with goat anti-mouse Alexa-647 (Thermo Scientific), and analyzed by flow cytometry. The results are shown in Figure 8. All Bb factor mAbs were able to inhibit C3 deposition on RBCs, with M4 / M12 > M20 > M10 / M18 > M17.

[0343] IC for inhibition of AP activity by Bb factor mAb in all three assays 50 The values ​​were calculated using Prism software and are shown in Figure 9. The IC values ​​generated in the Wieslab assay may be due to variations in serum concentrations used in each assay or differences in the measurement of the evaluation parameters. 50 The values ​​were consistently higher in the Wieslab assay than in the hemolysis / C3b deposition assay. In particular, Bb factor-specific M17 was less effective in the Wieslab assay compared to the hemolysis assay (IC). 50 A 17.7-fold difference; other mAbs showed IC50 between the two assays. 50 This showed a difference of 4.4 to 5.9 times. However, all assays reported the same relative efficacy among anti-Bb factor mAbs, which was inversely proportional to their specificity for Bb factor.

[0344] Figure 6. Inhibition of AP activity by anti-Bb factor mAbs.

[0345] Figure 7. Inhibition of AP pathway-mediated hemolysis by anti-Bb factor mAbs.

[0346] Figure 8. Inhibition of AP pathway-mediated C3b deposition by anti-Bb factor mAbs.

[0347] Figure 9. Comparison of the effect of factor Bb mAb on AP activity using three different assays. A nonlinear curve was fitted, and IC 50 This was calculated using Prism software.

[0348] Example 5: Inhibition of AP in cynomolgus monkeys by anti-Bb factor mAb The inhibition of complement alternative pathway (AP) activity by M10, an anti-Bb factor mAb, was measured using the Complement System Alternative Pathway WIESLAB® kit. Normal human serum or cynomolgus monkey ("cyno") serum (Innovative Research; 5.5%) was incubated with 2-fold serial dilutions of M10 or mouse isotype controls starting at 100 μg / mL. max The IC for M10 in this experiment was determined, and the results are shown in Figure 10. 50 The values ​​for human and cyno serum were 9.79E-8 and 3.67E-7M, respectively.

[0349] The inhibition of AP pathway-mediated hemolysis by anti-Bb factor mAbs was determined using cynomolgus monkey serum and rabbit erythrocytes in a buffer containing EGTA, which inhibits the classical pathway. Anti-Bb factor mAbs or mouse isotype controls (2-fold dilutions starting at 100 μg / mL) were incubated with 5% cynomolgus monkey serum and rabbit RBCs at 37°C for 1 hour. The supernatant A 540nm The amount of lysis was determined by measuring and subtracting the background absorbance in the control well containing EDTA. The results are shown in Figure 11. M17 does not inhibit cynoserum-mediated hemolysis of rabbit erythrocytes. IC against cynoserum 50 The values ​​were as follows: 2.497E-8 (M4), 2.202E-7 (M10), 2.824E-8 (M12), 2.319E-7 (M18), and 7.524E-7M (M20).

[0350] Figure 10. Inhibition of Wieslab AP activity in human or monkey serum by anti-Bb factor mAbs.

[0351] Figure 11. Inhibition of cyno-AP pathway-mediated hemolysis of rabbit RBCs by anti-Bb factor mAb.

[0352] Example 6: Sequencing of anti-Bb factor mAbs Amino acid sequencing of the VH and VL regions of factor Bb mAb was performed using techniques known to those skilled in the art (LakePharma). Specifically, cell pellets were prepared from hybridoma cell lines and RNA was extracted. The V region was amplified by reverse transcription polymerase chain reaction (RT-PCR) using a degenerate primer pool against a mouse antibody signal sequence along with constant-region primers for IgMVH, IgGVH, IgκVL, and IgλVL. The polymerase chain reaction (PCR) products obtained from each successful amplification were purified, cloned into the "TA" cloning vector system, and then sequenced. The putative amino acid sequences of the VH and VL regions of factor Bb mAB, as well as the CDR, are provided in Figures 12A-12F. M10 and M18 VL and VH are identical sequences, and M4 and M12 VH and VL are identical sequences.

[0353] Example 7: Further characterization of anti-Bb mAbs Inhibition of AP pathway-mediated hemolysis by Bb factor antibody in human RBCs pretreated with CD55 / CD59 antibody (Figure 14). Patients with paroxysmal nocturnal hemoglobinuria (PNH) suffer from complement-mediated lysis of their red blood cells (RBCs) due to a deficiency of complement regulators (CD55 and CD59) on the cell surface. Pretreatment with neutralizing antibodies against CD55 and CD59 makes healthy RBCs susceptible to complement-mediated lysis.

[0354] Normal human erythrocytes pretreated with anti-CD55 and CD59 antibodies were treated with various concentrations of Bb factor antibodies M4, M10, M17, and M20, as well as mouse IgG2a (isotype control). Cells were incubated with 20% normal human serum containing ) ). The reaction buffer contained 10 mM Mg EGTA to block the classical and lectin complement pathways. After 120 minutes at 37°C, the cells were centrifuged. The supernatant A 540nm The amount of lysis was determined by measuring the absorbance and subtracting the background absorbance in the control well containing EDTA. The results are shown in Figure 14. Complete inhibition of hemolysis was achieved only with the M10 antibody.

[0355] Production of anti-Bb factor chimeric antibodies Using the DNA sequence from Example 6, variable domains for hybridoma clones M4 and M10 were synthesized along with additional adjacent sequences required for cloning (DNA2.0). The variable domains were cloned into an expression vector, which generated chimeric antibodies composed of the mouse variable region and the human IgG4 constant / Fc region. The constructs were transfected into HEK293 cells and purified from the culture supernatant using Protein A Sepharose. The chimeric antibody containing the variable region of M10 is referred to as "Chimera M10". The chimeric antibody containing the variable region of M4 is referred to as "Chimera M4".

[0356] Inhibition of the complement replacement pathway (AP) by a Bb factor chimeric antibody (Figure 15) The inhibition of complement AP activity by chimeric M10 and M4 of Bb factor chimeric antibodies was measured using the Complement System Alternative Pathway WIESLAB® kit. 11% normal human serum (NHS; Complement Technologies) was incubated with 2-fold serial dilutions of chimeric antibodies or their parental monoclonal mouse antibodies starting at 300 μg / ml. A405 was measured at the end of the assay. The data are shown in Figure 15. A405 was proportional to the amount of final membrane invasion complex (MAC) deposited on the plate. A405 was plotted against antibody concentration. Chimeric antibodies were found to have comparable potency in the Wieslab Alternative Pathway assay compared to their corresponding parental monoclonal mouse antibodies.

[0357] Inhibition of AP pathway-mediated hemolysis in RBCs derived from patients with paroxysmal nocturnal hemoglobinuria (PNH) by a Bb factor chimeric antibody (Figure 16). Human erythrocytes derived from PNH patients were incubated with 20% O+ human serum containing various concentrations of chimeric M10 (human / mouse chimeric M10), anti-human C5 antibody, or human IgG4 (isotype control). The reaction buffer contained 10 mM Mg EGTA to block the classical and lectin complement pathways. After 180 minutes at 37°C, the cells were centrifuged. The supernatant A 540nm The amount of lysis was determined by measuring the background absorbance in the control well containing EDTA and subtracting it. The results are shown in Figure 16. Complete inhibition of hemolysis was achieved only with the chimeric M10 antibody.

[0358] Pharmacokinetics of Bb factor chimeric antibodies in cynomolgus monkeys (Figure 17) To determine the pharmacokinetic characteristics of Bb factor antibodies in cynomolgus monkeys, chimeric M10 and chimeric M4 were intravenously injected into three monkeys at 30 mg / kg, respectively. Plasma samples were collected for analysis at various time points after injection. Free chimeric M10 or chimeric M4 in diluted plasma samples were captured with Bb factor (fBb) protein on highly binding enzyme immunoassay (EIA) microtiter plates. The captured antibodies were detected with goat anti-human IgG secondary antibody conjugated with horseradish peroxidase (HRP) enzyme. The microtiter plates were washed to remove any unbound reactants, and then tetramethylbenzidine (TMB) substrate was reacted with immobilized HRP to produce a chromogenic product. The absorbance of this chromogenic product at a wavelength of 450 nm was directly proportional to the concentration of chimeric M10 or chimeric M4 in the sample. Data from ELISA were processed using GraphPad. The analysis was performed using the XY plot function and the 4-parameter logistic (4PL) linear regression function in Prism software to determine the presence of free chimeric M10 at each time point after drug administration. Alternatively, the amount of chimeric M4 was determined. The data are shown in Figure 17.

[0359] Pharmacodynamics of Bb factor chimeric antibodies in cynomolgus monkeys (Figures 18 and 19) To determine the pharmacodynamic properties of Bb factor antibodies in cynomolgus monkeys, chimeric M10 and chimeric M4 were intravenously injected into three monkeys at 30 mg / kg, respectively. Serum samples were collected for analysis at various time points after injection. Complement alternative pathway (CAP) and classical pathway (CCP) activity of serum samples was determined using the Complement System Alternative Pathway and Classical Pathway WIESLAB® kits, respectively, according to the manufacturer's recommended instructions. Serum concentrations of 5.5% and 0.99%, respectively, were used in the alternative and classical pathway assays. The graphs provided in Figures 18 and 19 are normalized to serum activity before antibody injection. As shown in Figure 18, chimeric M10 inhibits CAP activity but not CCP activity. As shown in Figure 19, chimeric M4 inhibits CAP activity and, to a lesser extent, CCP activity.

[0360] Chimeric M4 induces C3 degradation in human serum in vitro (Figure 20). Chimeric M4 was added to normal human serum at a concentration of 0.1 mg / ml and incubated at 37°C. Samples were collected at various time points for Western blotting analysis using rabbit anti-human C3 polyclonal antibody. Buffer was added to the serum as a negative control. Cobra venom factor (CVF), a protein known to induce C3 degradation, was added to normal human serum as a positive control. As shown in Figure 20, chimeric M4 induced C3 degradation in a time-dependent manner, similar to cobra venom factor.

[0361] Chimeric M4 induces C3 degradation in cynomolgus monkeys in vivo (Figure 21). Cynomolgus monkey serum samples from 30 mg / kg injections of chimeric M10 and chimeric M4 were assayed for C3 levels using sandwich ELISA. Briefly, purified goat anti-human C3 antibody was coated onto high-binding ELISA plates. After blocking, the plates were incubated with plasma samples collected at various time points after injection of chimeric M10 and chimeric M4 into monkeys. After washing, the plates were then incubated with biotinylated rabbit anti-human C3a antibody. After additional washing, the plates were incubated with streptavidin-horseradish peroxidase (HRP). Microtiter plates were washed to remove any unbound reactants, and then tetramethylbenzidine (TMB) substrate was reacted with immobilized HRP to produce a chromogenic product. The absorbance of this chromogenic product at a wavelength of 450 nm was directly proportional to the concentration of C3 in the sample. As shown in Figure 21 (upper panel), no change in C3 levels was observed with the injection of chimeric M10. As shown in Figure 21 (lower panel), a rapid loss of C3 was observed with the injection of chimeric M4.

[0362] The B / Bb factor antibody M4 blocks the DAF (degradation acceleration factor) activity of factor H (Figure 22). The effect of the B / Bb factor antibody M4 on the dissociation rate of C3 convertase by factor H (FH) was investigated using the Octet system (Pall ForteBio). Briefly, biotinylated propagine was prepared and bound to a streptavidin-coated Octet probe. The bound propagine probe was then incubated in a C3 convertase-containing solution prepared by mixing C3b, factor B (FB), and factor D (FD). Association was measured as the C3 convertase bound to the propagine probe. The probe was then transferred to a solution containing only buffer, and the dissociation of C3 convertase from the propagine-bound probe was measured. As shown in Figure 22, when factor H was added during the dissociation step, rapid dissociation of C3 convertase from the propagine probe could be measured (upper right). As shown in Figure 22, when C3 convertase was prepared in the presence of M4, rapid dissociation of C3 convertase by factor H was not observed.

[0363] The B / Bb factor antibody M4 does not block the DAF activity of CD55 (Figure 23). The effect of B / Bb factor antibody M4 on the CD55-mediated dissociation rate of C3 convertase was investigated using the Octet system (Pall ForteBio). Briefly, biotinylated propagine was prepared and bound to streptavidin-coated Octet probes. The bound propagine probes were then incubated in a solution containing C3 convertase (with bound B / Bb factor antibody M4) prepared by mixing C3b, factor B, B / Bb factor antibody M4, and factor D. Association was measured as the C3 convertase (with bound antibody) bound to the propagine probe. The probes were then transferred to a solution containing only buffer, and the dissociation of C3 convertase (with bound antibody) from the propagine-bound probe was measured. As shown in Figure 23, when CD55 was added during the dissociation step, rapid dissociation of C3 convertase (with bound antibody) from the propagine probe could be measured (below). The data indicates that binding of B / Bb factor antibodies to C3 convertase does not block the ability of CD55 to dissociate the C3 convertase complex.

[0364] Although the present invention is described in relation to its specific embodiments, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt specific situations, materials, compositions of substances, processes, or one or more steps of a process to the object, spirit, and scope of the invention. All such modifications are intended to fall within the scope of the claims appended herein.

Claims

[Claim 1] The invention as shown in the drawings.

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