Antigen binding molecules, pharmaceutical compositions and methods

Abstract

The present invention provides an antigen-binding molecule that binds to C1s and comprises a heavy chain variable region, a light chain variable region and an Fc region wherein (i) the heavy chain variable region and / or the light chain variable region comprises at least one amino acid wherein the antigen-binding molecule binds to C1s and wherein (i) the heavy chain variable region and / or the light chain variable region comprises at least one amino acid as compared to a first reference antibody, the at least one amino acid can increase the ratio of the KD value of the antigen-binding molecule to C1s in the acidic pH range to the KD value of the antigen-binding molecule to C1s in the neutral pH range, KD (acidic pH range) / KD (neutral pH range); (ii) the Fc region comprises at least one amino acid wherein the at least one amino acid can increase the binding capacity of the antigen-binding molecule to FcRn over an acidic pH range as compared to a second reference antibody; and (iii) the Fc region comprises at least one amino acid wherein the at least one amino acid can increase the binding capacity of the antigen-binding molecule to the Fc gamma receptor within a neutral pH range as compared to the second reference antibody.

Description

[0001] This application is a divisional application of a patent application with an application date of May 14, 2020, Chinese patent application number 202080048851.7, and invention name “Antigen Binding Molecules, Pharmaceutical Compositions and Methods”. Technical Field

[0002] The present invention relates to antigen binding molecules, pharmaceutical compositions and methods. Background Art

[0003] The complement system contains about 25-30 complement proteins that play a key role in host defense against pathogens, foreign antigens, and tumor cells. The complement system is also involved in maintaining homeostasis by clearing the body's immune complexes and apoptotic cells. The complement components perform these functions by interacting in a series of cascades of enzymatic processes and membrane-bound events. The end result of these processes is the production of products with lytic, immunomodulatory, and opsonizing functions.

[0004] As is well known, the complement system can be divided into three different pathways: the classical pathway, the lectin pathway, and the alternative pathway. Although each pathway has a different initiation, all three pathways converge and share the same terminal complement components (C5 to C9), which are ultimately responsible for destroying target cells.

[0005] The C1 complex is a large protein complex that has the function of being a key initiator of the classical pathway cascade. The C1 complex consists of three components (C1q, C1r and C1s), and the molar ratio of these three components is 1:2:2 (Non-patent Document 1). When the C1 complex binds to a target bound to an antibody, the classical pathway is initiated. C1q has six globular heads that mediate the binding of the C1 complex to the antibody through avidity interactions with the Fc region. Once tightly bound to the target, the C1r in the C1 complex is automatically activated and becomes enzymatically active. Then, the activated C1r cuts and activates the zymogen C1s in the C1 complex (Non-patent Document 2). Subsequently, the activated C1s cuts its substrate complement components C2 and C4 into C2a / C2b and C4a / C4b fragments, respectively. This results in the assembly of C4b2a (C3 convertase) on the target surface, which cuts C3 to form C3b. C3b sequentially cleaves C5 to initiate the formation of the terminal membrane attack complex C5b, C6, C7, C8, and C9, which lyse the target via pore formation.

[0006] C1s forms homodimers in a calcium-dependent manner (Non-patent Document 3). It is reported that at a calcium ion concentration of 1 mM, most of C1s is in a dimer state, while at a calcium ion concentration of 1 nM, it is mainly in a monomer state (Non-patent Document 4). In the circulation, C1s and C1r are mostly combined together as a calcium-dependent C1r2s2 heterotetramer, which in turn reversibly binds to C1q in a 1:1 ratio to form a C1 complex. In the absence or low concentration of calcium, the C1r2s2 tetramer dissociates into a C1r dimer and two C1s monomers (Non-patent Document 5). C1s is a 79kDa glycoprotein, 5% to 6% of its mass can be attributed to glycosylation (Non-patent Document 6). The C1s concentration in serum is reported to be about 55 μg / mL (0.7 μM) (Non-patent Document 7).

[0007] While a properly functioning complement system can defend the host against pathogens, dysregulation or inappropriate activation of the classical pathway can lead to various complement-mediated disorders, such as, but not limited to, autoimmune hemolytic anemia (AIHA), Behcet's disease, bullous pemphigus (BP), immune thrombocytopenic purpura (ITP), etc. Therefore, inhibiting excessive or uncontrolled activation of the classical pathway may provide clinical benefits to patients suffering from such disorders.

[0008] Antibodies are attractive drugs because they are stable in plasma, have high specificity for their targets, and generally exhibit favorable pharmacokinetic profiles. However, due to their large molecular size, the dose of therapeutic antibodies is generally high. In cases where the target is present in high abundance, the required antibody therapeutic dose is even higher. Therefore, methods to improve the pharmacokinetics, pharmacodynamics, and antigen-binding properties of antibodies are attractive approaches to reduce the dose and high production costs associated with therapeutic antibodies.

[0009] Some previous reports describe anti-C1s antibodies. For example, Matsumoto et al. (1986) (Non-Patent Document 8) described three antibodies that bind to different epitopes on C1. One clone showed preferential binding to the active form of C1s, while the other two bound to the zymogen and active C1s. Of these two clones, only one was able to inhibit C2 and C4 cleavage by C1s. Patent Document 1 describes antibodies that inhibit C1s-mediated C4 cleavage but not C2 cleavage. In addition, Patent Document 2 describes several antibodies that bind to a conformational epitope on C1s and are selective for active C1s compared to its zymogen form. Patent Document 3 describes two clones of anti-C1s antibodies that are capable of blocking C4 cleavage.

[0010] The affinity of antibody to its antigen determines the efficiency of antibody neutralization and its target. Various affinity maturation methods (non-patent literature 9) are used to increase antibody affinity to reduce the dosage required for therapeutic effect. However, limitation is that an antibody molecule usually has two binding sites, so only two targets (one antigen per binding site) are neutralized after administration. Even if antibody can bind target with infinite affinity by covalent interaction, the upper limit of the maximum target number of antibody neutralization remains at two.

[0011] It has been reported that antibodies that bind to antigens in a pH-dependent manner (hereinafter also referred to as "pH-dependent antibodies" or "pH-dependent binding antibodies") enable a single antibody molecule to neutralize multiple antigen molecules (Non-Patent Document 10, Patent Document 4). pH-dependent antibodies strongly bind to their antigens under neutral pH conditions in plasma, but dissociate from the antigen under acidic pH conditions in endosomes. Once dissociated from the antigen, the antibody is recycled back to the plasma by the FcRn receptor, and the dissociated antigen is degraded in the lysosomes of the cell. The recycled antibody can then freely bind to and neutralize the antigen molecule again, and the process continues to repeat as long as the antibody remains in circulation.

[0012] Reference List

[0013] Patent Literature

[0014] [PTL 1] WO2014 / 071206

[0015] [PTL 2] WO2014 / 066744

[0016] [PTL 3] WO2014 / 186599

[0017] [PTL 4] WO2009 / 125825

[0018] Non-patent literature

[0019] [NPL 1] Wang et al. Mol Cell. 2016 Jul 7;63(1):135-45

[0020] [NPL 2] Mortensen et al. Proc Natl Acad Sci US A. 2017 Jan 31;114(5):986-991

[0021] [NPL 3] Arlaud et al. Biochim Biophys Acta. 1980 Nov 6;616(1):105-15

[0022] [NPL 4] Rivas et al. Biochemistry. 1992 Dec 1;31(47):11707-12

[0023] [NPL 5] Rossi et al. Methods Mol Biol. 2014;1100:43-60

[0024] [NPL 6] Petillot et al. FEBS Lett. 1995 Jan 30;358(3):323-8

[0025] [NPL 7] Shi et al. Blood. 2014 Jun 26;123(26):4015-22

[0026] [NPL 8] Matsumoto et al. J Immunol. 1986 Nov 1;137(9):2907-12

[0027] [NPL 9] Kim et al. Methods Mol Biol. 2014;1131:407-20

[0028] [NPL 10] Igawa et al. Nat Biotechnol. 2010 Nov;28(11):1203-7 Summary of the invention

[0029] Technical issues

[0030] The present invention provides antigen binding molecules, pharmaceutical compositions and methods.

[0031] Solution to the problem

[0032] The present invention provides an antigen-binding molecule that binds to C1s and comprises a heavy chain variable region, a light chain variable region and an Fc region. The heavy chain variable region and / or the light chain variable region at least comprise specific amino acids that can change the antigen-binding ability in a specific manner. The Fc region at least comprises specific amino acids that can change its function in a specific manner.

[0033] Specifically, the present invention relates to:

[0034] [1] An antigen-binding molecule that binds to C1s and comprises a heavy chain variable region, a light chain variable region, and an Fc region, wherein

[0035] (i) the heavy chain variable region and / or the light chain variable region comprises at least one amino acid, wherein the at least one amino acid is capable of increasing the ratio of the KD value of the antigen-binding molecule with C1s in the acidic pH range to the KD value of the antigen-binding molecule with C1s in the neutral pH range, KD (acidic pH range) / KD (neutral pH range), compared to a first reference antibody comprising two heavy chain variable regions and two light chain variable regions, wherein the heavy chain variable region in the first reference antibody comprises the amino acid sequence of SEQ ID NO: 12 and the light chain variable region in the first reference antibody comprises the amino acid sequence of SEQ ID NO: 13;

[0036] (ii) the Fc region comprises at least one amino acid, wherein the at least one amino acid is capable of increasing the ability of the antigen-binding molecule to bind to FcRn in an acidic pH range compared to a second reference antibody comprising a paired Fc region, wherein the Fc region in the second reference antibody comprises the amino acid sequence of SEQ ID NO: 14; and

[0037] (iii) the Fc region comprises at least one amino acid, wherein the at least one amino acid can increase the binding ability of the antigen-binding molecule to the Fcγ receptor in the neutral pH range compared to the second reference antibody.

[0038] [2] The antigen-binding molecule of [1], wherein the heavy chain variable region and the light chain variable region comprise at least one amino acid that can increase the stability of the antigen-binding molecule compared to the first reference antibody.

[0039] [3] The antigen-binding molecule of [1] or [2], wherein the antigen-binding molecule comprises a constant region, wherein the Fc region of the antigen-binding molecule is in the constant region and the constant region comprises at least one amino acid that can reduce the immunogenicity of the antigen-binding molecule.

[0040] [4] The antigen-binding molecule of any one of [1] to [3], wherein the Fc region of the antigen-binding molecule comprises at least one amino acid that can reduce binding activity to rheumatoid factor.

[0041] [5] The antigen-binding molecule of any one of [1] to [4], wherein the variable region in (i) comprises at least one of the following amino acids;

[0042] The histidine at position 27 in the heavy chain variable region,

[0043] Proline at position 59 of the heavy chain variable region and

[0044] Histidine at position 96 of the light chain variable region

[0045] (All numbering is according to the Kabat numbering system).

[0046] [6] The antigen-binding molecule of any one of [1] to [5], wherein the Fc region in (ii) comprises at least one amino acid selected from the group consisting of leucine at position 428, alanine at position 434, and threonine at position 436 (all numbers are according to the EU numbering system).

[0047] [7] The antigen-binding molecule of any one of [1] to [6], wherein the Fc region in (iii) comprises at least one of the following amino acids;

[0048] Tyrosine at position 234,

[0049] Tryptophan at position 235,

[0050] Asparagine at position 236,

[0051] Aspartic acid at position 238,

[0052] Valine at position 250,

[0053] Isoleucine at position 264,

[0054] Aspartic acid at position 268,

[0055] Leucine at position 295,

[0056] Proline at position 307,

[0057] Threonine at position 326 and

[0058] Lysine at position 330

[0059] (All numbers are according to the EU numbering system).

[0060] [8] The antigen-binding molecule of any one of [1] to [7], wherein the Fc region in (iii) comprises the amino acids of the following (a) or (b);

[0061] (a) tryptophan at position 235, asparagine at position 236, aspartic acid at position 268, leucine at position 295, threonine at position 326, and lysine at position 330, or

[0062] (b) tyrosine at position 234, aspartic acid at position 238, valine at position 250, isoleucine at position 264, proline at position 307, and lysine at position 330

[0063] (All numbers are according to the EU numbering system).

[0064] [9] The antigen-binding molecule of [2], wherein the at least one amino acid capable of increasing stability is one or both of alanine at position 96 of the heavy chain variable region and glutamic acid at position 53 of the light chain variable region (both numberings are according to the Kabat numbering system).

[0065]

[10] The antigen-binding molecule of [3], wherein the at least one amino acid capable of reducing immunogenicity is arginine at position 214 according to the EU numbering system.

[0066]

[11] The antigen-binding molecule of [4], wherein the at least one amino acid capable of reducing the binding activity to rheumatoid factor is one or both of arginine at position 438 and glutamic acid at position 440 (both numberings are according to the EU numbering system).

[0067]

[12] An antigen-binding molecule that competes with an antibody for binding to C1s, the antibody comprising: a heavy chain variable region comprising HVR-H1 of SEQ ID NO: 20, HVR-H2 of SEQ ID NO: 21, and HVR-H3 of SEQ ID NO: 22, and a light chain variable region comprising HVR-L1 of SEQ ID NO: 23, HVR-L2 of SEQ ID NO: 24, and HVR-L3 of SEQ ID NO: 25.

[0068]

[13] The antigen-binding molecule of any one of [1] to

[12] , wherein the heavy chain variable region and the light chain variable region comprise a human framework.

[0069]

[14] An antigen-binding molecule comprising one or both of a VH region comprising 95% identity with the amino acid sequence of SEQ ID NO: 18 and a VL region comprising 95% identity with the amino acid sequence of SEQ ID NO: 19.

[0070]

[15] An antigen-binding molecule comprising: a heavy chain variable region comprising HVR-H1 of SEQ ID NO: 20, HVR-H2 of SEQ ID NO: 21, and HVR-H3 of SEQ ID NO: 22, and one or both of a light chain variable region comprising HVR-L1 of SEQ ID NO: 23, HVR-L2 of SEQ ID NO: 24, and HVR-L3 of SEQ ID NO: 25.

[0071]

[16] An antigen-binding molecule that binds to C1s and does not compete with C1s for binding to C1q.

[0072]

[17] The antigen-binding molecule of

[16] , which is the antigen-binding molecule of any one of [1] to

[15] .

[0073]

[18] An antigen-binding molecule whose epitope is located within the CCP-SP domain of C1s.

[0074]

[19] The antigen-binding molecule according to claim

[18] , which is the antigen-binding molecule according to any one of [1] to

[17] .

[0075]

[20] An antigen binding molecule that binds to C1s and is capable of reducing the plasma C1q concentration in a subject to 20% or less of the subject's baseline.

[0076]

[21] The antigen-binding molecule of

[20] , which is the antigen-binding molecule of any one of [1] to

[19] .

[0077]

[22] The antigen-binding molecule of

[20] or

[21] , wherein the individual is a cynomolgus monkey.

[0078]

[23] The antigen-binding molecule of any one of

[20] to

[22] , wherein the dose of the antigen-binding molecule is 10 mg / kg.

[0079]

[24] A pharmaceutical composition comprising the antigen-binding molecule of any one of [1] to

[23] and a pharmaceutically acceptable carrier.

[0080]

[25] A pharmaceutical composition of

[24] for use in treating a subject suffering from a complement-mediated disease or disorder.

[0081]

[26] A method for taking up C1s or C1q into cells, wherein the C1s and the cells are mixed in an isotonic solution, the cells express Fcγ receptors, and the method comprises adding the antigen-binding molecule of any one of [1] to

[23] to the isotonic solution.

[0082]

[27] A method for reducing the plasma C1s and / or C1q concentration in an individual, comprising administering the antigen-binding molecule of any one of [1] to

[23] to the individual.

[0083]

[28] A method for treating an individual suffering from a complement-mediated disease or disorder, the method comprising administering to the individual an effective amount of the antigen-binding molecule of any one of [1] to

[23] . BRIEF DESCRIPTION OF THE DRAWINGS

[0084] [ Figure 1 ] Figure 1BIACORE (registered trademark) sensorgrams showing the interaction of COS0098bb with human C1r2s2 and cynomolgus monkey C1r2s2 to evaluate pH dependence and cross-reactivity to cynomolgus monkey and human C1r2s2 (as described in Example 4.1) are shown. Sensorgrams were obtained by injecting human C1r2s2 (solid line) and cynomolgus monkey C1r2s2 (dashed line) on the sensor surface immobilized with COS0098bb, respectively. The antibody / antigen complex was allowed to dissociate at pH 7.4, followed by further dissociation at pH 5.8 (conditions indicated by arrows) to evaluate pH-dependent interactions.

[0085] [ Figure 2 ] Figure 2 BIACORE (registered trademark) sensorgrams showing the interaction of COS0098pHv1 with human C1r2s2 and cynomolgus monkey C1r2s2 to evaluate pH dependence and cross-reactivity against cynomolgus monkey and human C1r2s2 (as described in Example 4.1) are shown. Sensorgrams were obtained by injecting human C1r2s2 (solid line), cynomolgus monkey C1r2s2 (dashed line) on the sensor surface immobilized with COS0098pHv1, respectively. The antibody / antigen complex was allowed to dissociate at pH 7.4, followed by further dissociation at pH 5.8 (conditions indicated by arrows) to evaluate pH-dependent interactions.

[0086] [ Figure 3 ] Figure 3 is a graph showing the elimination of plasma C1s (Example 5) by COS0098pHv1-SG1077.

[0087] [ Figure 4 ] Figure 4 is a graph showing the elimination of plasma Clq by COS0098pHv1-SG1077 (Example 5). DETAILED DESCRIPTION

[0088] The techniques and procedures described or referenced herein are generally well understood by those skilled in the art and are commonly employed using conventional methodology, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd ed. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Modern Methods in Molecular Biology (FM Ausubel, et al., eds. (2003)); Enzymology Methods Series (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)), Harlow and Lane, eds. (1988); Antibodies, Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notes (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press. Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths and DG Newell, eds., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); PCR: Polymerase Chain Reaction, (Mullis et al., eds., 1994); Contemporary Immunological Methods (JE Coligan et al., eds., 1991); Molecular Biology in Brief (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott, Inc., 1993).

[0089] I. Definitions

[0090] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd edition, J. Wiley & Sons (New York, New York 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structures 4th edition, John Wiley & Sons (New York, New York 1992), provide general guidance for those skilled in the art for many of the terms used in this application. All references cited herein (including patent applications and publications) are incorporated by reference in their entirety.

[0091] For the purpose of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural, and vice versa. It should be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting. If any of the definitions listed below conflict with any document incorporated herein by reference, the definitions listed below shall prevail.

[0092] "Acceptor human framework" for the purposes of this article is a framework comprising an amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can include the same amino acid sequence, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0093] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X to its partner Y can generally be represented by a dissociation constant (Kd or KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below. The term "binding activity" refers to the strength of the sum of non-covalent interactions between a single or multiple binding sites of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Therefore, binding activity is not strictly limited to reflecting the activity of 1:1 interactions between members of a binding pair (e.g., an antibody and an antigen). When members of a binding pair can bind to each other in a monovalent and multivalent binding manner, the binding activity is the strength of the sum of these bindings. The binding activity of a molecule X to its partner Y can generally be represented by a dissociation constant (KD). Alternatively, the association rate and dissociation rate (Kon and Koff) can be used to assess binding. Binding activity can be measured by common methods known in the art, including the methods described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0094] An "affinity matured" antibody is one that has one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such alterations (such alterations resulting in an improvement in the affinity of the antibody for antigen).

[0095] The terms "anti-C1s antibody" and "antibody that binds to C1s" refer to an antibody that is capable of binding to C1s with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent targeting C1s. In one embodiment, the extent of binding of the anti-C1s antibody to unrelated, non-C1s proteins is less than about 10% of the binding of the antibody to C1s, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the antibody binds to C1s with a dissociation constant (Kd) of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M). In certain embodiments, an anti-C1s antibody binds to a C1s epitope that is conserved between C1s from different species.

[0096] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0097] "Antibody fragment" refers to a molecule other than an intact antibody but comprising a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0098] An "antibody that binds to the same epitope as a reference antibody" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

[0099] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0100] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major antibody classes: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0101] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 211 At 131 I. 125 I. 90 Y. 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P. 212radioactive isotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof, such as nucleases; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

[0102] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0103] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0104] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound to an antibody that targets the antigen, and includes specific amino acids that directly contact the antibody. Epitope determinants can include chemically active surface groupings of molecules (e.g., amino acids, sugar side chains, phosphoryl or sulfonyl groups), and can have specific three-dimensional structural features and / or specific charge characteristics. Typically, antibodies that are specific for a particular target antigen will preferentially recognize epitopes on the target antigen in a complex mixture of proteins and / or macromolecules.

[0105] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) in the Fc region may or may not exist. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0106] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain is typically composed of four FR domains: FR1, FR2, FR3, and FR4. Thus, HVR and FR sequences typically appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0107] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that comprise an Fc region as defined herein.

[0108] The terms "host cell", "host cell line" and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", including the transformed primary cell and the progeny derived therefrom, without regard to the number of passages. The progeny may not be completely identical to its parent cell in nucleic acid content and may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the originally transformed cell are included herein.

[0109] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or derived from a non-human source utilizing a human antibody library or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

[0110] "People's common framework" is a framework representing the most common amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is such as Kabat et al., Immunology Target Protein Sequences, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), a subgroup in Volumes 1-3. In one embodiment, for VL, the subgroup is such as subgroup κI in Kabat et al. (supra). In one embodiment, for VH, the subgroup is such as subgroup III in Kabat et al. (supra).

[0111] "Humanized" antibodies refer to chimeric antibodies comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one (and typically two) variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of non-human antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to a humanized antibody.

[0112] As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is hypervariable in sequence ("complementarity determining region" or "CDR") and / or forms structurally defined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contacts"). Typically, an antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0113] (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0114] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Protein Sequences of Immunological Targets, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0115] (c) antigenic contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and

[0116] (d) a combination of (a), (b) and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0117] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0118] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

[0119] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates (e.g., monkeys)), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0120] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0121] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0122] "Isolated nucleic acid encoding an anti-C1s antibody" refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0123] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody group, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or mutations produced during the production of monoclonal antibody preparations, such variants are usually present in small amounts), the individual antibodies that make up the group are identical and / or bind to the same epitope. Contrary to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the modifier "monoclonal" represents the characteristics of an antibody obtained from a substantially homogeneous antibody group, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibody to be used according to the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0124] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

[0125] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains connected by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of its constant domain, the light chain of an antibody can be designated as one of two types (called κ and λ).

[0126] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings concerning the use of such therapeutic products.

[0127] "Amino acid sequence identity percentage (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a reference polypeptide sequence after aligning the sequences and introducing gaps (if necessary) to achieve maximum sequence identity percentage and not considering any conservative substitutions as part of sequence identity. Alignment for purposes of determining amino acid sequence identity percentage can be achieved in various ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the sequence to be compared.

[0128] The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office in Washington, D.C., 20559, with user documentation, and the copyright registration number in the United States is TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. in South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program without change. In the case of using ALIGN-2 to compare amino acid sequences, the amino acid sequence identity % (optionally, it can be expressed as a given amino acid sequence A with, to, or for a given amino acid sequence B having or containing a specific amino acid sequence identity %) of a given amino acid sequence A is calculated as follows:

[0129] Multiply 100 by the X / Y fraction

[0130] Where X is the number of amino acid residues scored as identical matches in the program alignment of A and B by the sequence alignment program ALIGN-2, and Where Y is the total number of amino acid residues in B. It will be appreciated that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity between A and B will not be equal to the percent amino acid sequence identity between B and A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the preceding paragraph.

[0131] The term "pharmaceutical formulation" refers to a preparation that is in such form as to render the biological activity of the active ingredient contained therein effective, and that contains no additional components that would be unacceptably toxic to a subject to which the preparation would be administered.

[0132] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation that is non-toxic to a subject other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0133] Unless otherwise indicated, the term "C1s" as used herein refers to any native C1s from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed C1s as well as any form of C1s produced by processing in cells. The term also encompasses naturally occurring C1s variants, such as splice variants or allelic variants. The amino acid sequence of an exemplary human C1s is shown in SEQ ID NO: 1. The amino acid sequences of exemplary cynomolgus monkey and rat C1s are shown in SEQ ID Nos: 3 and 2, respectively.

[0134] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be used for prevention or performed during clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or relieving the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay disease development or slow disease progression.

[0135] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, the VH or VL domains from antibodies that bind to an antigen can be used to screen libraries of complementary VL or VH domains, respectively, to isolate antibodies that bind to a specific antigen. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0136] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0137] II. Antigen Binding Molecules

[0138] The antigen-binding molecule of the present invention binds to C1s and comprises a heavy chain variable region, a light chain variable region, and an Fc region.

[0139] In one embodiment, the heavy chain variable region and / or the light chain variable region comprises at least one amino acid, wherein the at least one amino acid is capable of increasing the ratio of the KD value of the antigen binding molecule to C1s in the acidic pH range to the KD value of the antigen binding molecule to C1s in the neutral pH range, KD (acidic pH range) / KD (neutral pH range), compared to a first reference antibody comprising two heavy chain variable regions and two light chain variable regions. In one embodiment, the heavy chain variable region in the first reference antibody comprises the amino acid sequence of SEQ ID NO: 12 and the light chain variable region in the first reference antibody comprises the amino acid sequence of SEQ ID NO: 13.

[0140] In this embodiment, the heavy chain variable region and / or the light chain variable region comprises a histidine at one or more of the following Kabat numbering system positions:

[0141] Heavy chain: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101, and H102; and

[0142] Light chain: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56 L91, L92, L93, L94, L95, L95a, L96 and L97.

[0143] In some embodiments, the heavy chain variable region and / or the light chain variable region comprises a histidine at one or more of the following Kabat numbering system positions:

[0144] Heavy chain: H26, H27, H28, H29, H30, H32, H33, H34, H50, H51, H52a, H54, H57, H58, H59, H60, H61, H65, H93, H95, H99, H100, and H100a; and

[0145] Light chain: L25, L28, L91, L92, L94, L95, L96 and L97.

[0146] In some embodiments, the heavy chain variable region and / or the light chain variable region comprises at least one histidine substituted for one or more amino acid residues at a position selected from the following Kabat numbering system positions:

[0147] Heavy chain: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52, H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94, H95, H96, H97, H98, H99, H100, H100a, H101, and H102; and

[0148] Light chain: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56 L91, L92, L93, L94, L95, L95a, L96 and L97.

[0149] In some embodiments, any one or more amino acids of the heavy chain variable region and / or light chain variable region provided above are substituted with histidine at the following Kabat numbering system positions:

[0150] Heavy chain: H51, H65 and H99; and

[0151] Light chain: L92, L94, L95 and L96.

[0152] In some embodiments, the heavy chain variable region and / or the light chain variable region comprises one, two, three, four, or five histidines replacing amino acid residues at the following Kabat numbering system positions:

[0153] Heavy chain: H51, H65 and H99; and

[0154] Light chain: L92, L94, L95 and L96.

[0155] In some embodiments, the heavy chain variable region and / or the light chain variable region comprises at least one histidine, which is a substituted residue at one or more of the following positions and a CDR or FR amino acid position (according to the Kabat numbering system):

[0156] Heavy chain: H51, H65 and H99; and

[0157] Light chain: L92, L94, L95 and L96.

[0158] In some embodiments, the heavy chain variable region and / or the light chain variable region comprises at least one histidine which is the substituted residue at the following positions (according to the Kabat numbering system):

[0159] 1) L92 and L94

[0160] 2) L92 and L95

[0161] 3) L94 and L95

[0162] 4) L92, L94 and L95

[0163] 5) H65 and L92

[0164] 6) H65 and L94

[0165] 7) H65 and L95

[0166] 8) H65, L92 and L94

[0167] 9) H65, L92 and L95

[0168] 10) H65, L94 and L95

[0169] 11) H65, L92, L94 and L95

[0170] 12) H99 and L92

[0171] 13) H99 and L94

[0172] 14) H99 and L95

[0173] 15) H99, L92 and L94

[0174] 16) H99, L92 and L95

[0175] 17) H99, L94 and L95

[0176] 18) H99, L92, L94 and L95

[0177] 19) H65 and H99

[0178] 20) H65, H99 and L92

[0179] 21) H65, H99 and L94

[0180] 22) H65, H99 and L95

[0181] 23) H65, H99, L92 and L94

[0182] 24) H65, H99, L92 and L95

[0183] 25) H65, H99, L94 and L95

[0184] 26) H65, H99, L92, L94 and L95, or

[0185] 27) H27, H99 and L95.

[0186] In this embodiment, the variable region preferably comprises at least one of the following amino acids: histidine at position 27 of the heavy chain variable region, proline at position 59 of the heavy chain variable region, and histidine at position 96 of the light chain variable region (all numbering is according to the Kabat numbering system).

[0187] In one embodiment, the Fc region comprises at least one amino acid, wherein the at least one amino acid is capable of increasing the binding ability of the antigen binding molecule to FcRn in the acidic pH range compared to a second reference antibody comprising a paired Fc region. In this embodiment, the Fc region in the second reference antibody comprises the amino acid sequence of SEQ ID NO: 14.

[0188] In this embodiment, the Fc region comprises

[0189] (a) according to EU numbering, Ala at position 434; Glu, Arg, Ser or Lys at position 438; and Glu, Asp or Gln at position 440;

[0190] (b) according to EU numbering, Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440;

[0191] (c) according to EU numbering, Ile or Leu at position 428; Ala at position 434; Ile, Leu, Val, Thr or Phe at position 436; Glu, Arg, Ser or Lys at position 438; and Glu, Asp or Gln at position 440;

[0192] (d) according to EU numbering, Ile or Leu at position 428; Ala at position 434; Ile, Leu, Val, Thr or Phe at position 436; Arg or Lys at position 438; and Glu or Asp at position 440;

[0193] (e) according to EU numbering, Leu at position 428; Ala at position 434; Val or Thr at position 436; Glu, Arg, Ser or Lys at position 438; and Glu, Asp or Gln at position 440; or

[0194] (f) According to EU numbering, Leu at position 428; Ala at position 434; Val or Thr at position 436; Arg or Lys at position 438; and Glu or Asp at position 440.

[0195] WO2013 / 046704 specifically reports that double amino acid residue substitutions according to EU numbering Q438R / S440E, Q438R / S440D, Q438K / S440E and Q438K / S440D, when combined with amino acid substitutions capable of increasing FcRn binding under acidic conditions, result in a significant reduction in rheumatoid factor binding.

[0196] In this embodiment, the Fc region preferably comprises a combination of amino acid substitutions selected from the group consisting of:

[0197]

[0198]

[0199] under the EU number; or

[0200]

[0201] According to EU numbering.

[0202] In another embodiment, the Fc region preferably comprises at least one amino acid selected from the group consisting of leucine at position 428, alanine at position 434, and threonine at position 436 (all numbering is according to the EU numbering system). In this embodiment, the Fc region more preferably comprises leucine at position 428, alanine at position 434, and threonine at position 436 (all numbering is according to the EU numbering system).

[0203] In one embodiment, the Fc region comprises at least one amino acid that increases the ability of the antigen binding molecule to bind to an Fcγ receptor in the neutral pH range compared to a second reference antibody.

[0204] In this embodiment, the Fc region preferably comprises at least one or more amino acids selected from the group consisting of:

[0205] According to EU numbering, in the Fc region site

[0206] Lys or Tyr at amino acid position 221;

[0207] any one of Phe, Trp, Glu and Tyr at amino acid position 222;

[0208] any one of Phe, Trp, Glu and Lys at amino acid position 223;

[0209] any one of Phe, Trp, Glu and Tyr at amino acid position 224;

[0210] any one of Glu, Lys and Trp at amino acid position 225;

[0211] any one of Glu, Gly, Lys and Tyr at amino acid position 227;

[0212] any one of Glu, Gly, Lys and Tyr at amino acid position 228;

[0213] any one of Ala, Glu, Gly and Tyr at amino acid position 230;

[0214] any one of Glu, Gly, Lys, Pro and Tyr at amino acid position 231;

[0215] any one of Glu, Gly, Lys and Tyr at amino acid position 232;

[0216] any one of Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 233;

[0217] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 234;

[0218] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 235;

[0219] any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 236;

[0220] any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 237;

[0221] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 238;

[0222] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 239;

[0223] any one of Ala, Ile, Met and Thr at amino acid position 240;

[0224] any one of Asp, Glu, Leu, Arg, Trp and Tyr at amino acid position 241;

[0225] any one of Leu, Glu, Leu, Gln, Arg, Trp and Tyr at amino acid position 243;

[0226] His at amino acid position 244;

[0227] Ala at amino acid position 245;

[0228] any one of Asp, Glu, His and Tyr at amino acid position 246;

[0229] any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val and Tyr at amino acid position 247;

[0230] any one of Glu, His, Gln and Tyr at amino acid position 249;

[0231] Glu or Gln at amino acid position 250;

[0232] Phe at amino acid position 251;

[0233] any one of Phe, Met and Tyr at amino acid position 254;

[0234] any one of Glu, Leu and Tyr at amino acid position 255;

[0235] any one of Ala, Met and Pro at amino acid position 256;

[0236] any one of Asp, Glu, His, Ser and Tyr at amino acid position 258;

[0237] any one of Asp, Glu, His and Tyr at amino acid position 260;

[0238] any one of Ala, Glu, Phe, Ile and Thr at amino acid position 262;

[0239] any one of Ala, Ile, Met and Thr at amino acid position 263;

[0240] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 264;

[0241] any one of Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 265;

[0242] any one of Ala, Ile, Met and Thr at amino acid position 266;

[0243] any one of Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 267;

[0244] any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val and Trp at amino acid position 268;

[0245] any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 269;

[0246] any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp and Tyr at amino acid position 270;

[0247] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 271;

[0248] any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 272;

[0249] Phe or Ile at amino acid position 273;

[0250] any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 274;

[0251] Leu or Trp at amino acid position 275;

[0252] any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 276;

[0253] any one of Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val and Trp at amino acid position 278;

[0254] Ala at amino acid position 279;

[0255] any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp and Tyr at amino acid position 280;

[0256] any one of Asp, Lys, Pro and Tyr at amino acid position 281;

[0257] any one of Glu, Gly, Lys, Pro and Tyr at amino acid position 282;

[0258] any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg and Tyr at amino acid position 283;

[0259] any one of Asp, Glu, Leu, Asn, Thr and Tyr at amino acid position 284;

[0260] any one of Asp, Glu, Lys, Gln, Trp and Tyr at amino acid position 285;

[0261] any one of Glu, Gly, Pro and Tyr at amino acid position 286;

[0262] any one of Asn, Asp, Glu and Tyr at amino acid position 288;

[0263] any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp and Tyr at amino acid position 290;

[0264] any one of Asp, Glu, Gly, His, Ile, Gln and Thr at amino acid position 291;

[0265] any one of Ala, Asp, Glu, Pro, Thr and Tyr at amino acid position 292;

[0266] any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 293;

[0267] any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 294;

[0268] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 295;

[0269] any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr and Val at amino acid position 296;

[0270] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 297;

[0271] any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 298;

[0272] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp and Tyr at amino acid position 299;

[0273] any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val and Trp at amino acid position 300;

[0274] any one of Asp, Glu, His and Tyr at amino acid position 301;

[0275] Ile at amino acid position 302;

[0276] any one of Asp, Gly and Tyr at amino acid position 303;

[0277] any one of Asp, His, Leu, Asn and Thr at amino acid position 304;

[0278] any one of Glu, Ile, Thr and Tyr at amino acid position 305;

[0279] any one of Ala, Asp, Asn, Thr, Val and Tyr at amino acid position 311;

[0280] Phe at amino acid position 313;

[0281] Leu at amino acid position 315;

[0282] Glu or Gln at amino acid position 317;

[0283] any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val and Tyr at amino acid position 318;

[0284] any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 320;

[0285] any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp and Tyr at amino acid position 322;

[0286] Ile at amino acid position 323;

[0287] any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 324;

[0288] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 325;

[0289] any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp and Tyr at amino acid position 326;

[0290] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp and Tyr at amino acid position 327;

[0291] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 328;

[0292] any one of Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 329;

[0293] any one of Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 330;

[0294] any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp and Tyr at amino acid position 331;

[0295] any one of Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr at amino acid position 332;

[0296] any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val and Tyr at amino acid position 333;

[0297] any one of Ala, Glu, Phe, Ile, Leu, Pro and Thr at amino acid position 334;

[0298] any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp and Tyr at amino acid position 335;

[0299] any one of Glu, Lys and Tyr at amino acid position 336;

[0300] any one of Glu, His and Asn at amino acid position 337;

[0301] any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser and Thr at amino acid position 339;

[0302] Ala or Val at amino acid position 376;

[0303] Gly or Lys at amino acid position 377;

[0304] Asp at amino acid position 378;

[0305] Asn at amino acid position 379;

[0306] any one of Ala, Asn and Ser at amino acid position 380;

[0307] Ala or Ile at amino acid position 382;

[0308] Glu at amino acid position 385;

[0309] Thr at amino acid position 392;

[0310] Leu at amino acid position 396;

[0311] Lys at amino acid position 421;

[0312] Asn at amino acid position 427;

[0313] Phe or Leu at amino acid position 428;

[0314] Met at amino acid position 429;

[0315] Trp at amino acid position 434;

[0316] Ile at amino acid position 436; and

[0317] Any one of Gly, His, Ile, Leu and Tyr at amino acid position 440.

[0318] In this embodiment, the Fc region more preferably comprises at least one or more of the following

[0319] According to EU numbering, in the Fc region site

[0320] Asp at amino acid position 238, and

[0321] Glu at amino acid position 328.

[0322] In this embodiment, the Fc region further preferably comprises at least one of the following amino acids: tyrosine at position 234, tryptophan at position 235, asparagine at position 236, aspartic acid at position 238, valine at position 250, isoleucine at position 264, aspartic acid at position 268, leucine at position 295, proline at position 307, threonine at position 326 and lysine at position 330 (all numbering is according to the EU numbering system). The Fc region further preferably comprises the following amino acids (a) or (b);

[0323] (a) tryptophan at position 235, asparagine at position 236, aspartic acid at position 268, leucine at position 295, threonine at position 326, and lysine at position 330, or

[0324] (b) tyrosine at position 234, aspartic acid at position 238, valine at position 250, isoleucine at position 264, proline at position 307 and lysine at position 330 (all numbering according to the EU numbering system).

[0325] In one embodiment, the isoelectric point (pI) of antigen binding molecules is increased by the change of Fc district. In this embodiment, compared with its parent Fc district, the antigen binding molecules with increased pI include at least two amino acid changes in the Fc district. In a further embodiment, compared with the isoelectric point (pI) in the parent Fc district, each amino acid change increases the isoelectric point (pI) in the Fc district. In a further embodiment, amino acid can be exposed on the surface of this region. In a further embodiment, antigen binding molecules include Fc district and antigen binding domains. In a further embodiment, the antigen binding activity of antigen binding domains changes according to ion concentration conditions.

[0326] In a further embodiment, the Fc region with increased pI of the present invention comprises at least two amino acid changes at at least two positions selected from the group consisting of: 285, 311, 312, 315, 318, 333, 335, 337, 341, 342, 343, 384, 385, 388, 390, 399, 400, 401, 402, 413, 420, 422 and 431 (according to EU numbering). In a further embodiment, the Fc region with increased pI comprises Arg or Lys at each selected position.

[0327] In a specific embodiment, the Fc region with increased pi comprises an arginine at position 311 and an arginine at position 343 (both numbering according to the EU numbering system).

[0328] In one embodiment, the heavy chain variable region and the light chain variable region comprise at least one amino acid that increases the stability of the antigen binding molecule compared to the first reference antibody. In this embodiment, one or both of the alanine at position 96 of the heavy chain variable region and the glutamic acid at position 53 of the light chain variable region (both numberings are according to the Kabat numbering system) are preferred as such amino acids.

[0329] In one embodiment, the antigen binding molecules include constant regions. In this embodiment, the Fc region is present in the constant region. In this embodiment, the constant region preferably includes at least one amino acid that can reduce the immunogenicity of the antigen binding molecules. In this embodiment, the arginine at position 214 (according to the EU numbering system) is preferably used as such amino acid.

[0330] In one embodiment, the Fc region of the antigen binding molecule comprises at least one amino acid capable of reducing the rheumatoid factor binding activity. In this embodiment, one or both of the arginine at position 438 and the glutamic acid at position 440 (both numberings are according to the EU numbering system) are preferably used as amino acids.

[0331] In one aspect, the antigen binding molecule binds to C1s, wherein the antigen binding molecule competes for binding to C1s with an antibody comprising: a heavy chain variable region comprising HVR-H1 of SEQ ID NO: 20, HVR-H2 of SEQ ID NO: 21, HVR-H3 of SEQ ID NO: 22, and a light chain variable region comprising HVR-L1 of SEQ ID NO: 23, HVR-L2 of SEQ ID NO: 24, and HVR-L3 of SEQ ID NO: 25. In this aspect, the antigen binding molecule may have the heavy chain variable region, light chain variable region, and Fc region in any of the above embodiments.

[0332] In one aspect, the antigen binding molecule binds to C1s and comprises one or both of a VH region comprising 95% identity with the amino acid sequence of SEQ ID NO: 18 and a VL region comprising 95% identity with the amino acid sequence of SEQ ID NO: 19. In this aspect, the antigen binding molecule may have the heavy chain variable region, light chain variable region, and Fc region in any of the above embodiments.

[0333] In one aspect, the antigen binding molecule binds to C1s and comprises: a heavy chain variable region comprising HVR-H1 of SEQ ID NO: 20, HVR-H2 of SEQ ID NO: 21, and HVR-H3 of SEQ ID NO: 22, and one or both of a light chain variable region comprising HVR-L1 of SEQ ID NO: 23, HVR-L2 of SEQ ID NO: 24, and HVR-L3 of SEQ ID NO: 25. In this aspect, the antigen binding molecule may have the heavy chain variable region, light chain variable region, and Fc region in any of the above embodiments.

[0334] In one aspect, the antigen binding molecule binds to C1s and does not compete with C1s for binding to C1q. In this aspect, the antigen binding molecule may have the heavy chain variable region, light chain variable region and Fc region in any of the above embodiments.

[0335] In one aspect, the antigen binding molecule binds to C1s, wherein its epitope is located within the CCP-SP domain of C1s. In this aspect, the antigen binding molecule may have the heavy chain variable region, light chain variable region and Fc region in any of the above embodiments.

[0336] On the one hand, the antigen binding molecule binds to C1s and can reduce the plasma C1q concentration of the individual to 20% or less of the individual baseline. In this regard, the antigen binding molecule may have a heavy chain variable region, a light chain variable region, and an Fc region in any of the above embodiments. In one embodiment, the individual is a cynomolgus monkey. In one embodiment, the dose of the antigen binding molecule is 10 mg / kg. In this embodiment, the antigen binding molecule can be intravenously injected into the monkey on day 0. The dose setting of the antigen binding molecule can be adjusted to an excess concentration of the physiological plasma cynomolgus monkey C1s and C1q concentrations immediately after injection. Blood can be collected at -1 day before injection and 5 minutes, 2, 8 hours, 1, 2, 4, 7, 14, 21, and 28 days after injection. The blood sample can be centrifuged immediately to separate the plasma sample. The plasma concentrations of cynomolgus monkey C1s and C1q can be measured at each sampling point by LC / ESI-MS / MS.

[0337] III. Compositions and Methods

[0338] In one aspect, the antigen binding molecules of the invention can be used, for example, in the diagnosis or treatment of a complement-mediated disease or disorder.

[0339] In one embodiment, examples of anti-C1s antibodies are the above-mentioned antigen-binding molecules. When any anti-C1s antibody is mentioned below, the anti-C1s antibody may be replaced with an antigen-binding molecule that binds to C1s.

[0340] A. Exemplary anti-C1s antibodies

[0341] In one aspect, the present invention provides an isolated antibody that binds to C1s. In one aspect, the present invention provides an isolated antibody that binds to C1s, whose binding activity varies according to ion concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies according to pH, i.e., hydrogen ion (proton) concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies according to calcium concentration. In certain embodiments, the binding activity of the anti-C1s antibody varies according to both pH and calcium concentration. Such antibodies are expected to be particularly advantageous as drugs because the dosage and frequency of administration to patients can be reduced, thereby reducing the total dosage.

[0342] In one aspect, in the isolated anti-C1s antibody of the present invention, the ratio of the KD value of the C1s binding activity at an acidic pH to the KD value of the C1s binding activity at a neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or more when measured at neutral and acidic pH with high calcium concentration. In one aspect, in the isolated anti-C1s antibody of the present invention, the ratio of its koff value of the C1s binding activity at an acidic pH to the koff value of the C1s binding activity at a neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or more when measured at neutral and acidic pH with high calcium concentration. On the one hand, in the isolated anti-C1s antibody of the present invention, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 5 or more when measured at neutral pH, high calcium concentration and at acidic pH, low calcium concentration. In some embodiments, in the isolated anti-C1s antibody of the present invention, the ratio of its KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or more when measured at neutral and acidic pH, low calcium concentration, wherein the anti-C1s antibody binds to the dimeric state of C1s. In some embodiments, in the isolated anti-C1s antibody of the present invention, the ratio of the koff value of its C1s binding activity at acidic pH to the koff value of its C1s binding activity at neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or greater when measured at neutral and acidic pH and low calcium concentration, wherein the anti-C1s antibody binds to the dimeric state of C1s.

[0343] Without being bound by a particular theory, in the following cases: 1) the epitope structure of C1s bound to the antibody of the present invention may undergo a conformational change due to the absence of calcium, thereby changing the affinity of the antibody or 2) the interaction (affinity or avidity) of the antibody of the present invention may vary depending on the state of C1s (monomer state or dimeric state), measurements using specific conditions (at neutral pH with high calcium concentration and at acidic pH with low calcium concentration) can be used to evaluate the ratio of KD values ​​(KD (acidic pH) / KD (neutral pH)).

[0344] In other words, as described in (i) or (iii) below, the antibodies of the present invention bind to C1s with a higher affinity at neutral pH than at acidic pH:

[0345] (i) when measured at neutral and acidic pH with high calcium concentration, the ratio of the KD value for C1s binding activity at acidic pH to the KD value for C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or more,

[0346] (ii) when measured at neutral and acidic pH with high calcium concentration, the ratio of the koff value of the C1s binding activity at acidic pH to the koff value of the C1s binding activity at neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or more,

[0347] (iii) when measured at a neutral pH with a high calcium concentration and at an acidic pH with a low calcium concentration, the ratio of the KD value for the C1s binding activity at an acidic pH to the KD value for the C1s binding activity at a neutral pH (KD(acidic pH) / KD(neutral pH)) is 5 or more.

[0348] More generally, without being bound by a particular theory, in the following situations: 1) the epitope structure of a specific antigen that binds to an antibody of the invention may undergo conformational changes due to the absence of calcium, thereby changing the affinity of the antibody or 2) the interaction (affinity or avidity) of an antibody of the invention may vary depending on the state of the antigen (monomer state or dimeric state), measurements using specific conditions (high calcium concentration at neutral pH and low calcium concentration at acidic pH) can be used to estimate the ratio of KD values ​​(KD (acidic pH) / KD (neutral pH)). If this ratio is high, the affinity at acidic pH is lower than the affinity at neutral pH. Alternatively, as described below, KD is defined as k off / k on The ratio between acidic and neutral conditions off The ratio of the values, ie, (koff(acidic pH) / koff(neutral pH)) can also be used to compare the affinity at acidic and neutral pH values.

[0349] Therefore, the antibodies of the present invention bind to the antigen with higher affinity at neutral pH than at acidic pH, as shown below: the ratio of the KD value for antigen-binding activity at acidic pH to the KD value for antigen-binding activity at neutral pH (KD (acidic pH) / KD (neutral pH)) is 5 or more when measured at neutral pH, high calcium concentration, and acidic pH, low calcium concentration.

[0350] On the one hand, in the isolated anti-C1s antibody of the present invention, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater when measured at neutral and acidic pH and high calcium concentrations.

[0351] On the one hand, in the isolated anti-C1s antibody of the present invention, the ratio of the KD value of its C1s binding activity at acidic pH to the KD value of its C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 5 or greater when measured at neutral pH with high calcium concentration and at acidic pH with low calcium concentration.

[0352] On the one hand, in the isolated anti-C1s antibody of the present invention, the ratio of the KD value of the C1s binding activity at acidic pH to the KD value of the C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater when measured at neutral and acidic pH and low calcium concentration, wherein the anti-C1s antibody binds to the dimeric state of C1s.

[0353] The above-mentioned KD ratio, i.e., KD (acidic pH) / KD (neutral pH), can be compared between a parent antibody (i.e., the original antibody before modification of the present invention) and an antibody into which one or more amino acid mutations (e.g., addition, insertion, deletion or substitution) have been introduced with respect to the original (parent) antibody. The original (parent) antibody may be any known or newly isolated antibody, as long as it specifically binds to C1s. Thus, in one aspect, the ratio of the KD value of the C1s binding activity at acidic pH values ​​to the KD value of the C1s binding activity at neutral pH values ​​(KD(acidic pH) / KD(neutral pH)) in the isolated anti-C1s antibody of the present invention is at least 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2-fold, 2.1-fold, 2,2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 8-fold, or 10-fold higher than the ratio of the KD value of the C1s binding activity at acidic pH values ​​to the KD value of the C1s binding activity at neutral pH values ​​(KD(acidic pH) / KD(neutral pH)) of the original (parent) antibody. In other words, the present invention provides an isolated anti-C1s antibody, wherein the isolated anti-C1s antibody has introduced one or more amino acid mutations (e.g., addition, insertion, deletion or substitution) compared to the parent (original) antibody, and the ratio of the following (i) to (ii) is at least 1.2, 1.4, 1.6, 1.8, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.5, 2.7, 2.8, 2.9, 3, 3.5, 4, 5, 8 or 10: (i) the ratio of the KD value of the C1s binding activity of the isolated anti-C1s antibody at an acidic pH value to the KD value of the C1s binding activity at a neutral pH value (KD(acidic pH) / KD(neutral pH)); (ii) the ratio of the KD value of the C1s binding activity of the parent (original) antibody at an acidic pH value to the KD value of the C1s binding activity at a neutral pH value (KD(acidic pH) / KD(neutral pH)). These KD ratios can be measured at any (high or low) calcium concentration, e.g., at neutral and acidic pH with high calcium concentration, or at neutral pH with high calcium concentration and at acidic pH with low calcium concentration. On the other hand, the dissociation rate constant (kd) can be used instead of the above KD to evaluate pH and / or Ca dependence.

[0354] On the one hand, the antibody of the present invention has different antigen binding activity between intracellular conditions and extracellular conditions. Intracellular and extracellular conditions refer to different conditions between the inside and outside of a cell. The categories of conditions include, for example, ion concentration, more specifically, metal ion concentration, hydrogen ion concentration (pH) and calcium ion concentration. "Intracellular conditions" preferably refer to an environment with characteristics of the internal environment, while "extracellular conditions" preferably refer to an environment with characteristics of the environment in plasma. By screening a large number of antibodies for a domain with such characteristics, antibodies with characteristics that antigen binding activity varies with ion concentration can be obtained. For example, antibodies with a large number of sequences different from each other are produced by a hybridoma method or an antibody library method, and their antigen binding activities under different ion concentrations are measured, and antibodies with the above characteristics can be obtained. The B cell cloning method is one of the method examples for screening such antibodies. In addition, as described below, at least one unique amino acid residue that can be assigned to an antibody with characteristics of antigen binding activity that varies according to ion concentration is prepared as a library of a large number of antibodies with different sequences and sharing different amino acid residues as a common structure. Such libraries can be screened to effectively separate antibodies with the above characteristics.

[0355] In one aspect, the present invention provides antibodies that bind to C1s with a higher affinity at neutral pH than at acidic pH. In another aspect, the present invention provides anti-C1s antibodies that exhibit pH-dependent binding to C1s. As used herein, the expression "pH-dependent binding" means "reduced binding at acidic pH compared to neutral pH," and the two expressions are interchangeable. For example, an anti-C1s antibody "having pH-dependent binding properties" includes an antibody that binds to C1s with a higher affinity at neutral pH than at acidic pH.

[0356] In certain embodiments, the ratio of the KD value for C1s binding activity at acidic pH to the KD value for C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater when measured at neutral and acidic pH with high calcium concentrations. In specific embodiments, the antibodies of the invention bind to C1s at neutral pH with an affinity that is at least 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times greater than at acidic pH.

[0357] In certain embodiments, the ratio of the koff value of C1s binding activity at acidic pH to the koff value of C1s binding activity at neutral pH (koff(acidic pH) / koff(neutral pH)) is 2 or greater when measured at neutral and acidic pH with high calcium concentration. In certain embodiments, an antibody of the invention binds to C1s at neutral pH with an affinity that is at least 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more times greater at acidic pH.

[0358] In certain embodiments, the ratio of the KD value for C1s binding activity at acidic pH to the KD value for C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 2 or greater when measured at neutral pH, high calcium concentration, and at acidic pH, low calcium concentration. In certain embodiments, the antibodies of the invention have an ATPase activity at a neutral pH that is at least 2, 3, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 200, 400, 1000, 10000 or more times greater affinity binds to C1s.

[0359] In the above case, for example, the acidic pH is 5.8 and the neutral pH is 7.4, so KD (acidic pH) / KD (neutral pH) is KD (pH 5.8) / KD (pH 7.4). In this regard, examples of acidic pH and neutral pH are described in detail below. In some embodiments, KD (acidic pH) / KD (neutral pH) such as KD (pH 5.8) / KD (pH 7.4) can be 2 to 10,000. In the above case, for example, the acidic pH is 5.8 and the neutral pH is 7.4, so koff (acidic pH) / koff (neutral pH) is koff (pH5.8) / koff (pH 7.4). In this regard, examples of acidic pH and neutral pH are described in detail below. In some embodiments, koff(acidic pH) / koff(neutral pH), for example, koff(pH 5.8) / koff(pH 7.4), may be 2 to 10,000.

[0360] When the antigen is a soluble protein, the binding of the antibody to the antigen can lead to an extension of the half-life of the antigen in plasma (i.e., a reduction in the clearance rate of the antigen from the plasma) because the half-life of the antibody in plasma is longer than that of the antigen itself and can serve as a carrier of the antigen. This is due to the recycling of the antigen-antibody complex by FcRn through the intracellular endosomal pathway (Roopenian and Akilesh (2007) Nat Rev Immunol 7 (9): 715-725). However, antibodies with pH-dependent binding properties (which bind to their antigen in a neutral extracellular environment while releasing the antigen into the acidic endosomal compartment after it enters the cell) are expected to have superior properties in terms of antigen neutralization and clearance relative to counterparts that bind in a pH-independent manner (Igawa et al. (2010) Nature Biotechnol 28(11); 1203-1207; Devanaboyina et al. (2013) mAbs 5(6): 851-859; International Patent Application Publication No.: WO 2009 / 125825).

[0361] In one aspect, the invention provides antibodies that bind to C1s with a higher affinity under conditions of high calcium concentration than under conditions of low calcium concentration.

[0362] In the present invention, preferred metal ions include, for example, calcium ions. Calcium ions are involved in the regulation of many biological phenomena, including contraction of muscles (e.g., skeletal muscle, smooth muscle, and cardiac muscle); activation of leukocyte movement, phagocytosis, etc.; activation of platelet morphological changes, secretion, etc.; activation of lymphocytes; activation of mast cells, including the secretion of histamine; cell reactions mediated by catecholamine α receptors or acetylcholine receptors; exocytosis; release of transmitter substances from neuronal terminals; and axoplasmic flow in neurons. Known intracellular calcium ion receptors include troponin C, calmodulin, parvalbumin, and myosin light chain, which have several calcium ion binding sites and are believed to be derived from a common origin in terms of molecular evolution. There are also many known calcium binding motifs. Such well-known motifs include, for example, cadherin domains, the EF hand of calmodulin, the C2 domain of protein kinase C, the Gla domain of the coagulation protein Factor IX, C-type lectins of the acyaroglycoprotein receptor and the mannose binding receptor, the A domain of the LDL receptor, annexins, type 3 thrombospondin domains, and EGF-like domains.

[0363] In the present invention, when the metal ion is a calcium ion, it is expected that the antigen binding activity under low calcium ion concentration conditions is lower than the antigen binding activity under high calcium ion concentration conditions. At the same time, the intracellular calcium ion concentration is lower than the extracellular calcium ion concentration. On the contrary, the extracellular calcium ion concentration is higher than the intracellular calcium ion concentration. In the present invention, the low calcium ion concentration is preferably 0.1 micromolar (μM) to 30 μM, more preferably 0.5 μM to 10 μM, and particularly preferably 1 μM to 5 μM, which is close to the calcium ion concentration of the early endosome in vivo. At the same time, in the present invention, the high calcium ion concentration is preferably 100 μM to 10 μM, more preferably 200 μM to 5 mM, and particularly preferably 0.5mM to 2.5mM, which is close to the calcium ion concentration in plasma (in blood). In the present invention, it is preferred that the low calcium ion concentration is the calcium ion concentration in the endosome, and the high calcium ion concentration is the calcium ion concentration in the plasma. When comparing the level of antigen binding activity between low calcium ion concentration and high calcium ion concentration, it is preferred that the antibody of the present invention has a stronger binding ratio at high calcium ion concentration than at low calcium ion concentration. In other words, it is preferred that the antibody of the present invention has a lower antigen binding activity at low calcium ion concentration than at high calcium ion concentration. When the binding activity level is represented by a dissociation constant (KD), the value of KD (low calcium ion concentration) / KD (high calcium ion concentration) is greater than 1, preferably 2 or greater, more preferably 10 or greater, and more preferably 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or greater. The upper limit of the value of KD (low calcium ion concentration) / KD (high calcium ion concentration) is not particularly limited, and can be any value such as 100, 400, 1000 or 10000, as long as the technician can produce using technology. Dissociation rate constant (kd) can be used instead of KD.When it is difficult to calculate KD value, activity can be assessed based on the binding response level in Biacore when analyte passes through with the same concentration.When antigen is by being fixed with the chip of antigen binding molecules of the present invention, the binding response under low calcium concentration is preferably 1 / 2 of the binding response under high calcium concentration or less, more preferably 1 / 3 or less, also more preferably 1 / 5 or less, and particularly preferably 1 / 10 or less.It is known that the extracellular calcium ion concentration (for example, in blood plasma) is high in common body, and the intracellular calcium ion concentration (for example, in endosome) is low.Therefore, in the present invention, it is preferred that the extracellular condition is high calcium ion concentration, and the intracellular condition is low calcium ion concentration.When giving the antigen binding molecules of the present invention (for example, antibody) with the lower property of antigen binding activity under intracellular calcium ion concentration condition than under extracellular calcium ion concentration condition, the antigen combined with the antigen binding molecules of the present invention in the extracellular is dissociated from the antigen binding molecules of the present invention in the cell.Such antibodies, when applied to living bodies, can reduce the antigen concentration in plasma and reduce the physiological activity of antigens in vivo. Therefore, the antibody of the present invention is useful. The method of screening the antigen-binding domains or antibodies with lower antigen-binding activity under low calcium ion concentration conditions than under high calcium ion concentration conditions includes, for example, the method described in WO2012 / 073992 (for example, the 0200-0213 paragraphs). The method of giving the antigen-binding domains of the present invention the property of being weaker than that under high calcium ion concentration conditions with antigen binding is not particularly limited, and can be carried out by any method. Specifically, the method is described in Japanese Patent Application No. 2011-218006 and includes, for example, at least one amino acid residue in the antigen-binding domains is replaced with an amino acid residue with metal chelating activity, and / or at least one amino acid residue with metal chelating activity is inserted into the antigen-binding domains. The antigen-binding molecules of the present invention in which at least one amino acid residue of the antigen-binding domain has been substituted with an amino acid residue having metal chelating activity and / or at least one amino acid residue having metal chelating activity has been inserted into the antigen-binding domain are preferred embodiments of the antigen-binding molecules of the present invention.

[0364] The amino acid residue with metal chelating activity preferably includes, for example, serine, threonine, asparagine, glutamine, aspartic acid and glutamic acid. In addition, the amino acid residue that changes the antigen-binding activity of antigen-binding domains according to calcium ion concentration preferably includes, for example, the amino acid residue that forms calcium binding motif. Calcium binding motifs are well known to those skilled in the art and have been described in detail (e.g., Springer et al., (Cell (2000) 102, 275-277); Kawasaki and Kretsinger (Protein Prof. (1995) 2, 305-490); Moncrief et al., (J. Mol. Evol. (1990) 30, 522-562); Chauvaux et al., (Biochem. J. (1990) 265, 261-265); Bairoch and Cox (FEBS Lett. (1990) 269, 454-456); Davis (New Biol. (1990) 2, 410-419); Schaefer et al., (Genomics (1995) 25, 638 to 643); Economou et al., (EMBO J. (1990) 9, 349-354); Wurzburg et al., (Structure. (2006) 14, 6, 1049-1058)). The EF hand of troponin C, calmodulin, parvalbumin and myosin light chain; the C2 domain in protein kinase C; the Gla domain of coagulation protein factor IX; the C-type lectins of acylglycoprotein receptors and mannose binding receptors, ASGPR, CD23 and DC-SIGN; the A domain of LDL receptor; the annexin domain; the cadherin domain; the type 3 thrombospondin domain; and the EGF-like domain are preferably used as calcium binding motifs.

[0365] The antigen-binding domains of the present invention may contain amino acid residues that change antigen-binding activity according to calcium ion concentration, such as the above-mentioned amino acid residues with metal chelating activity and amino acid residues that form calcium binding motifs. The positions of these amino acid residues in the antigen-binding domains are not particularly limited, and they can be located at any position, as long as the antigen-binding activity changes according to the calcium ion concentration. At the same time, such amino acid residues can be included alone or in a combination of two or more, as long as the antigen-binding activity changes according to the calcium ion concentration. The amino acid residues preferably include, for example, serine, threonine, asparagine, glutamine, aspartic acid and glutamic acid. When the antigen-binding domain is an antibody variable region, the amino acid residues may be included in the heavy chain variable region and / or the light chain variable region. In a preferred embodiment, the amino acid residues may be included in the CDR3 of the heavy chain variable region, more preferably in the CDR3 of the heavy chain variable region according to the 95th, 96th, 100a and / or 101st of Kabat numbering.

[0366] In another preferred embodiment, the amino acid residue may be contained in the CDR1 of the light chain variable region, more preferably in the CDR1 of the light chain variable region according to Kabat numbering at position 30, 31 and / or 32. In yet another preferred embodiment, the amino acid residue may be contained in the CDR2 of the light chain variable region, more preferably in the CDR2 of the light chain variable region according to Kabat numbering at position 50. In yet another preferred embodiment, the amino acid residue may be contained in the CDR3 of the light chain variable region, more preferably in the CDR3 of the light chain variable region according to Kabat numbering at position 92.

[0367] Furthermore, the above embodiments may be combined. For example, the amino acid residue may be contained in two or three CDRs selected from CDR1, CDR2 and CDR3 of the light chain variable region, more preferably at position 30, 31, 32, 50 and / or 92 according to Kabat numbering in the light chain variable region.

[0368] A large number of antigen-binding domains having different sequences while sharing the common structure of the above-mentioned amino acid residues that change antigen-binding activity according to calcium ion concentration are prepared as libraries. The library can be screened to efficiently obtain antigen-binding domains having binding activity to the desired antigen, wherein their antigen-binding activity changes according to calcium ion concentration.

[0369] For the purposes of the present disclosure, the "affinity" of an antibody for C1s is expressed as the antibody's KD. The KD of an antibody refers to the equilibrium dissociation constant of an antibody-antigen interaction. The greater the KD value of an antibody binding to its antigen, the weaker its binding affinity for a particular antigen. Therefore, as used herein, the expression "higher affinity at neutral pH than at acidic pH" (or the equivalent expression "pH-dependent binding") means that the KD of an antibody at an acidic pH is greater than the KD value of the antibody at a neutral pH. For example, in the context of the present invention, if the KD of an antibody binding to C1s at an acidic pH is at least 2 times greater than the KD of an antibody binding to C1s at a neutral pH, the antibody is considered to bind to C1s at a neutral pH with a higher affinity than at an acidic pH. Thus, the invention includes antibodies that bind to C1s at acidic pH with a KD that is at least 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times or more higher than at neutral pH. In another embodiment, the KD value of the antibody at neutral pH may be 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In another embodiment, the KD value of the antibody at an acidic pH value may be 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M or larger.

[0370] The binding properties of an antibody to a specific antigen can also be expressed by the antibody's kd. The antibody's kd refers to the dissociation rate constant of the antibody relative to the specific antigen, expressed in reciprocal seconds (i.e., sec -1). An increase in the kd value indicates that the antibody binds to its antigen less strongly. Thus, the invention includes antibodies that bind to C1s at acidic pH values ​​with a higher kd value than at neutral pH values. The invention includes antibodies that bind to C1s at acidic pH values ​​with a kd that is at least 2, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 times or more higher than the kd that the antibody binds to C1s at neutral pH values. In another embodiment, the antibody may have a kd value of 10 at neutral pH. -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 1 / s or less. In another embodiment, the kd value of the antibody at an acidic pH value may be 10 -3 1 / s, 10 -2 1 / s, 10 -1 1 / s or greater.

[0371] In some cases, "reduced binding at acidic pH values ​​compared to neutral pH values" is expressed as the ratio of the KD value of the antibody at acidic pH values ​​to the KD value of the antibody at neutral pH values ​​(or vice versa). For example, for the purposes of the present invention, an antibody can be considered to exhibit "reduced binding to C1s at acidic pH values ​​compared to its binding at neutral pH values" if the antibody exhibits an acidic / neutral KD ratio of 2 or greater. In certain exemplary embodiments, the acidic / neutral KD ratio of an antibody of the invention can be 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the KD value of the antibody at neutral pH can be 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12In another embodiment, the KD value of the antibody at an acidic pH value may be 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M or larger.

[0372] Alternatively, "reduced binding at acidic pH values ​​compared to neutral pH values" is expressed as the ratio of the antibody's koff value at acidic pH values ​​to the antibody's koff value at neutral pH values ​​(or vice versa). For example, for the purposes of the present invention, an antibody may be considered to exhibit "reduced binding to C1s at acidic pH values ​​compared to its binding at neutral pH values" if the antibody exhibits an acidic / neutral koff ratio of 2 or greater. In certain exemplary embodiments, the acidic / neutral koff ratio of an antibody of the invention can be 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more.

[0373] In some cases, "reduced binding at acidic pH values ​​compared to neutral pH values" is expressed as the ratio of the antibody's kd value at acidic pH values ​​to the antibody's kd value at neutral pH values ​​(or vice versa). For example, for the purposes of the present invention, an antibody can be considered to exhibit "reduced binding to C1s at acidic pH values ​​compared to its binding at neutral pH values" if it exhibits an acidic / neutral kd ratio of 2 or greater. In certain exemplary embodiments, the acidic / neutral kd ratio of the antibodies of the invention can be 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000 or more. In another embodiment, the kd value of the antibody at neutral pH can be 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 1 / s or less. In another embodiment, the kd value of the antibody at an acidic pH value may be 10 -31 / s, 10 -2 1 / s, 10 -1 1 / s or greater.

[0374] As used herein, the expression "acidic pH value" means a pH value of 4.0 to 6.5. The expression "acidic pH value" includes pH values ​​of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 and 6.5. In a specific aspect, the "acidic pH value" is 5.8 or 6.0.

[0375] As used herein, the expression "neutral pH value" means a pH value of 6.7 to about 10.0. The expression "neutral pH value" includes pH values ​​of 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 and 10.0. In a specific aspect, the "neutral pH value" is 7.0 or 7.4.

[0376] As used herein, the expression "under high calcium concentration conditions" or "at a high calcium concentration" means 100 μM to 10 mM, more preferably 200 μM to 5 mM, and particularly preferably 0.5 mM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood). The expression "under high calcium concentration conditions" or "at high calcium concentration" includes 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 0.5 mM, 0.7 mM, 0.9 mM, 1 mM, 1.2 mM, 1.4 mM, 1.6 mM, 1.8 mM, 2.0 mM, 2.2 mM, 2.4 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM and 10 mM Ca. 2+ In certain aspects, "under high calcium concentration conditions" or "at high calcium concentration" refers to 1.2 mM Ca 2 + .

[0377] As used herein, the expression "under low calcium concentration conditions" or "at a low calcium concentration" means 0.1 μM to 30 μM, more preferably 0.5 μM to 10 μM, and particularly preferably 1 μM to 5 μM, which is close to the calcium ion concentration of the early endosome in vivo. The expression "under low calcium concentration conditions" or "at a low calcium concentration" includes 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, 2.0 μM, 2.5 μM, 2.6 μM, 2.7 μM, 2.8 μM, 2.9 μM, 3.0 μM, 3.1 μM, 3.2 μM, 3.3 μM, 3.4 μM, 3.5 μM, 4.0 μM, 5.0 μM, 6.0 μM, 7.0 μM, 8.0 μM, 9.0 μM, 10 μM, 15 μM, 20 μM, 25 μM and 30 μM Ca 2+ In certain aspects, "under low calcium concentration conditions" or "at low calcium concentration" refers to 3.0 μM Ca 2+ .

[0378] KD values ​​and kd values ​​as expressed herein can be determined using a surface plasmon resonance-based biosensor to characterize antibody-antigen interactions. (See, e.g., Example 2 herein). KD values ​​and kd values ​​can be determined at 25 degrees Celsius (°C) or 37°C. The assay can be performed in the presence of 150 mM NaCl. In some embodiments, the assay can be performed using surface plasmon resonance technology, wherein the antibody is immobilized, the antigen is used as an analyte, and the following conditions are used: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate and 150 mM NaCl, 37 degrees Celsius (°C).

[0379] In one aspect, the present invention provides an anti-C1s antibody having pH dependence, wherein the antibody comprises at least one histidine in the variable region, wherein at least one amino acid in the variable region is replaced by another amino acid, such that

[0380] 1) The non-specific binding activity of the antibody will be reduced at acidic pH and / or neutral pH, or

[0381] 2) The ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) will increase.

[0382] In one aspect, the present invention provides an anti-C1s antibody having pH dependence, wherein the antibody comprises at least one histidine in the variable region, wherein at least one amino acid in the variable region is substituted with an amino acid selected from the group consisting of D, E, K, R and Q, such that

[0383] 1) The non-specific binding activity of the antibody will be reduced at acidic pH and / or neutral pH, or

[0384] 2) The ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) will increase.

[0385] In a certain aspect, the expression "non-specific binding activity" means the extracellular matrix (ECM) binding activity of the antibody. In a certain aspect, the expression "non-specific binding activity" means the ECM binding activity of the antibody at acidic pH values. In certain embodiments, at least one amino acid in the anti-C1s antibody of the present invention may be substituted with one or more amino acids such that the ECM binding activity is reduced at acidic pH values. In certain embodiments, at least one amino acid in the anti-C1s antibody of the present invention may be substituted with one or more amino acids such that the ECM binding activity is reduced at neutral pH values.

[0386] On the one hand, the present invention provides an anti-C1s antibody with pH dependence, wherein at least one amino acid is substituted in the variable region, thereby reducing the ECM binding activity of the antibody. In certain embodiments, the ECM binding activity is reduced at acidic pH values. In certain embodiments, the ECM binding activity is reduced at neutral pH values. In certain embodiments, such antibodies refer to antibodies having one or more changes in one or more hypervariable regions (HVRs), such changes resulting in an improvement in the ECM binding activity of the antibody to the antigen, i.e., a reduction in the ECM binding activity, compared to a parent antibody without such changes.

[0387] The method for measuring "extracellular matrix binding" is not particularly limited, and can be measured using an ELISA system, which detects the binding between the polypeptide and the extracellular matrix by adding the polypeptide to the extracellular matrix immobilization plate and adding a labeled antibody for the polypeptide. In particular, the measurement method using the electrochemiluminescence (ECL) method is preferred because the method can detect the extracellular matrix binding ability with higher sensitivity. Specifically, a mixture of a polypeptide and a ruthenium antibody is added to the extracellular matrix fixed plate, and the binding between the polypeptide and the extracellular matrix can be measured using an ECL system that measures ruthenium electrochemiluminescence. The added polypeptide concentration can be set arbitrarily, and preferably a high concentration is added to improve the detection sensitivity of the extracellular matrix binding. Although the extracellular matrix used in the present invention can be plant-derived or animal-derived, as long as it contains glycoproteins, such as collagen, proteoglycans, fibronectin, laminin, endogenous power, fibrin and basement membrane proteoglycan, the present invention preferably has an extracellular matrix of animal origin; and for example, an extracellular matrix of animal (such as human, mouse, rat, monkey, rabbit and dog) origin can be used. Particularly, in order to monitor the improvement of human plasma dynamics, preferably naturally occurring human extracellular matrix derived from people. In addition, the condition for evaluating polypeptide and extracellular matrix combination is expected to be in the neutral range (physiological condition) close to pH 7.4, but not necessarily in the neutral range, and can also be evaluated in the acidic range (close to pH 6.0). In addition, when evaluating the combination of polypeptide and extracellular matrix, the antigen molecule combined with polypeptide can be made to coexist to evaluate the combination of polypeptide / antigen molecule complex and extracellular matrix.

[0388] In some embodiments, whether the non-specific binding activity of an antibody at an acidic pH is reduced can be measured by using, for example, ELISA or ECL. In a further embodiment, in the isolated anti-C1s antibody of the present invention, the ECM binding value between the parent antibody (i.e., the original antibody before D, E, K, R and / or Q substitution) and the antibody into which one amino acid substitution (D, E, K, R and / or Q) relative to the original (parent) antibody has been introduced can be compared, provided that the antibody contains at least one histidine in the variable region. The original (parent) antibody can be any known or newly isolated antibody as long as it specifically binds to C1s. Therefore, in one aspect, in the isolated anti-C1s antibody of the present invention, the ECM binding value of the replaced antibody is at least 1.2 times, 1.4 times, 1.6 times, 1.8 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 5 times, 8 times, 10 times lower than the ECM binding value of the original (parent) antibody.

[0389] Without being limited by a particular theory, the histidine residues in the antibody can interact with various residues around the histidine residues in the antibody. This interaction can affect the structure of the antibody or the conformation of the CDR. Histidine is protonated and positively charged at acidic pH values. Introducing positively charged residues (such as arginine or lysine) at the position around the histidine can result in repulsion between the positively charged residues and the protonated histidine at acidic pH values, thereby inducing the structure or conformational changes of the antibody or CDR. Similarly, introducing negatively charged residues (such as aspartic acid or glutamic acid) at the position around the histidine can result in interaction between the negatively charged residues and the protonated histidine at acidic pH values, thereby inducing the structure or conformational changes of the antibody or CDR. These structural or conformational changes of the antibody or CDR that occur at acidic pH values ​​can affect the antigen binding of the antibody, and reduce the binding affinity of the antibody to the antigen at acidic pH values. In summary, the introduction of charged residues (such as arginine, lysine, aspartic acid or glutamic acid) around the histidine residues in antibodies can reduce the binding affinity of antibodies to antigens at acidic pH, thereby increasing the pH dependence of antibody-antigen interactions by a unique mechanism.

[0390] In one aspect, the present invention provides a method for increasing the ratio of the KD value of the antigen-binding activity of an antibody at an acidic pH to the KD value of the antigen-binding activity at a neutral pH (KD(acidic pH) / KD(neutral pH)), comprising:

[0391] 1) Providing an antibody having pH dependence comprising at least one histidine in the variable region,

[0392] 2) replacing at least one amino acid in the antibody variable region with an amino acid selected from the group consisting of D, E, K, R, Q and H.

[0393] In one aspect, the present invention provides a method for enhancing the clearance (or removal) of an antigen from plasma, comprising

[0394] 1) Providing an antibody having pH dependence comprising at least one histidine in the variable region,

[0395] 2) replacing at least one amino acid in the antibody variable region with an amino acid selected from the group consisting of D, E, K, R, Q and H.

[0396] In one aspect, the present invention provides a method for promoting antigen binding molecule-mediated antigen uptake into cells, comprising:

[0397] 1) Providing an antibody having pH dependence comprising at least one histidine in the variable region,

[0398] 2) replacing at least one amino acid in the antibody variable region with an amino acid selected from the group consisting of D, E, K, R, Q and H.

[0399] In one aspect, the invention provides a method for increasing the number of antigens that can be bound by a single antigen-binding molecule, comprising:

[0400] 1) Providing an antibody having pH dependence comprising at least one histidine in the variable region,

[0401] 2) replacing at least one amino acid in the antibody variable region with an amino acid selected from the group consisting of D, E, K, R, Q and H.

[0402] In one aspect, the present invention provides a method for enhancing the ability of an antigen-binding molecule to eliminate an antigen from plasma, comprising:

[0403] 1) Providing an antibody having pH dependence comprising at least one histidine in the variable region,

[0404] 2) replacing at least one amino acid in the antibody variable region with an amino acid selected from the group consisting of D, E, K, R, Q and H.

[0405] In certain embodiments, in the above methods of the present invention, the distance between the histidine residue comprised by the variable region and the replaced amino acid (i.e., D, E, K, R, Q or H) is less than 20 angstroms, 18 angstroms, 16 angstroms, 14 angstroms, 12 angstroms, 10 angstroms, 8 angstroms, 6 angstroms, 4 angstroms or 2 angstroms.

[0406] In one aspect, the present invention provides a method for increasing the clearance of C1s from the plasma of an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C1s antibody of the present invention to increase the clearance of C1s from the plasma. The present invention also provides a method for increasing the clearance of C1r and C1s complexes from the plasma of an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C1s antibody of the present invention to increase the clearance of C1r and C1s complexes from the plasma. The present invention also provides a method for increasing the clearance of C1q, C1r and C1s complexes from the plasma of an individual. In some embodiments, the method comprises administering to the individual an effective amount of an anti-C1s antibody of the present invention to increase the clearance of C1q, C1r and C1s complexes from the plasma.

[0407] In another aspect, the present invention provides a method for removing C1s from plasma, the method comprising: (a) identifying an individual in need of removing C1s from the individual's plasma; (b) providing an antibody that binds to C1s via the antigen binding (C1s binding) domain of the antibody and has a KD (pH 5.8) / KD (pH 7.4) value, wherein the KD (pH 5.8) / KD (pH 7.4) value is defined as the ratio of the KD for C1s at pH 5.8 to the KD for C1s at pH 7.4 when the KD is determined using surface plasmon resonance technology, which is 2 to 10,000, wherein the antibody binds to C1s in plasma in vivo and dissociates from the bound C1s under conditions present in endosomes in vivo, and wherein the antibody is a human IgG or a humanized IgG; and (c) administering the antibody to the individual. In yet another aspect, this surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In yet another aspect, a surface plasmon resonance technique can be used in which the antibody is immobilized, the antigen is used as the analyte, and the following conditions are used: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl at 37° C. In yet another aspect, the dissociation rate constant (kd) can be used instead of the above KD.

[0408] In another aspect, the present invention provides a method for removing C1s from plasma of a subject, the method comprising: (a) identifying a first antibody that binds to C1s through an antigen binding domain of the first antibody; (b) identifying a second antibody that: (1) binds to C1s through an antigen binding (C1s binding) binding domain of the second antibody, (2) has an amino acid sequence identical to the first antibody, except that at least one amino acid in the variable region of the first antibody is replaced by histidine and / or at least one histidine is inserted into the variable region of the first antibody, and (3) has a KD(pH5.8) / KD(pH7.4) value that is higher than the KD(pH5.8) / KD(pH7.4) value of the first antibody and is between 2 and 10,000, wherein KD(pH5.8) / KD(pH7.4) is defined as the ratio of the KD for C1s at pH 5.8 to the KD for C1s at pH 7.4 when the KD is determined using surface plasmon resonance technology. 7.4, (4) binds to C1s in plasma in vivo, (5) dissociates from bound C1s under conditions present in endosomes in vivo, and (6) is human IgG or humanized IgG; (c) identifying a subject in need of reduction of plasma C1s levels; and (d) administering a second antibody to the subject to reduce the plasma C1s levels of the subject. In yet another aspect, this surface plasmon resonance technique can be used at 37°C and 150 mM NaCl. In yet another aspect, this surface plasmon resonance technique can be used at 37°C and 150 mM NaCl. In yet another aspect, such a surface plasmon resonance technique can be used in which the antibody is immobilized, the antigen is used as the analyte, and the following conditions are used: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl at 37°C. In yet another aspect, the dissociation rate constant (kd) can be used instead of the above KD.

[0409] In another aspect, the present invention provides a method for removing C1s from plasma of a subject, the method comprising: (a) identifying a first antibody that: (1) binds to C1s via an antigen binding domain of the first antibody, (2) is identical in amino acid sequence to a second antibody that binds to C1s via an antigen binding (C1s binding) domain of a second antibody, except that at least one variable region of the first antibody has at least one more histidine residue than the corresponding variable region of the second antibody, and (3) has a KD(pH5.8) / KD(pH7.4) value that is higher than the KD(pH5.8) / KD(pH7.4) value of the second antibody and is between 2 and 10,000, wherein KD(pH5.8) / KD(pH7.4) is defined as the KD for C1s at pH 5.8 and the KD for C1s at pH 7.4 when the KD is determined using surface plasmon resonance technology. 7.4 for C1s, (4) binds to C1s in plasma in vivo, (5) dissociates from bound C1s under conditions present in endosomes in vivo, and (6) is human IgG or humanized IgG; (b) identifying a subject in need of reduction of plasma C1s levels; and (c) administering at least one first antibody to the subject, thereby reducing the plasma C1s levels of the subject. In another aspect, such surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In another aspect, such surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In another aspect, such surface plasmon resonance technology can be used, wherein the antibody is immobilized, the antigen is used as the analyte, and the following conditions are used: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate and 150 mM NaCl at 37°C. The present invention also provides a method for inhibiting the cleavage of complement component C4, wherein the antibody does not inhibit the cleavage of complement component C2. In certain instances, the antibody inhibits a component of the classical complement pathway; in certain instances, the classical complement pathway component is Cls. In yet another aspect, the dissociation rate constant (kd) can be used in place of the KD described above.

[0410] On the one hand, the present disclosure provides a method for regulating complement activation. In some embodiments, the method inhibits complement activation, for example to reduce the production of C4b2a. In some embodiments, the present disclosure provides a method for regulating complement activation in an individual suffering from a complement-mediated disease or condition, the method comprising administering to the individual an anti-C1s antibody of the present disclosure or a pharmaceutical composition of the present disclosure, wherein the pharmaceutical composition comprises an anti-C1s antibody of the present disclosure. In some embodiments, this method inhibits complement activation. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. Administration can be by any route known to those skilled in the art, including those disclosed herein. In some embodiments, administration is intravenous administration. In some embodiments, administration is intrathecal administration.

[0411] In certain embodiments, the anti-C1s antibodies of the invention bind to C1s from more than one species. In specific embodiments, the anti-C1s antibodies bind to C1s from humans and non-human animals. In specific embodiments, the anti-C1s antibodies bind to C1s from humans, rats, and monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees, and baboons).

[0412] In some embodiments, anti-C1s antibody variants are provided, which are prepared by introducing amino acid modifications into antibodies VH1 / Vk1, VH1 / Vk2, VH1 / Vk3, VH2 / Vk1, VH2 / Vk2, VH2 / Vk3, VH3 / Vk1, VH3 / Vk2, VH3 / Vk3, VH4 / Vk1, VH4 / Vk2 or VH4 / Vk3 disclosed in WO2014 / 071206.

[0413] In some embodiments, an anti-C1s antibody of the invention comprises a histidine at one or more of the following Kabat numbering system positions:

[0414] Heavy chain: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52,H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94,H95, H96, H97, H98, H99, H100, H100a, H101 and H102; and

[0415] Light chains: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56 L91, L92, L93, L94, L95, L95a, L96 and L97.

[0416] In some embodiments, an anti-C1s antibody of the invention comprises a histidine at one or more of the following Kabat numbering system positions:

[0417] Heavy chain: H26, H27, H28, H29, H30, H32, H33, H34, H50, H51, H52a, H54,H57, H58, H59, H60, H61, H65, H93, H95, H99, H100 and H100a; and

[0418] Light chain: L25, L28, L91, L92, L94, L95, L96 and L97.

[0419] In some embodiments, an anti-C1s antibody of the invention comprises at least one histidine replacing one or more amino acid residues at a position selected from the following Kabat numbering system positions:

[0420] Heavy chain: H26, H27, H28, H29, H30, H31, H32, H33, H34, H35, H50, H51, H52,H52a, H53, H54, H55, H57, H58, H59, H60, H61, H62, H63, H64, H65, H93, H94,H95, H96, H97, H98, H99, H100, H100a, H101 and H102; and

[0421] Light chains: L24, L25, L26, L27, L27a, L28, L29, L30, L31, L32, L33, L50, L51, L52, L53, L54, L55, L56 L91, L92, L93, L94, L95, L95a, L96 and L97.

[0422] In some embodiments, any one or more amino acids of an anti-C1s antibody provided above are substituted with histidine at the following Kabat numbering system positions:

[0423] Heavy chain: H51, H65 and H99; and

[0424] Light chain: L92, L94, L95 and L96.

[0425] In some embodiments, an isolated anti-C1s antibody of the invention comprises one, two, three, four, or five histidines substituted for amino acid residues at the following Kabat numbering system positions:

[0426] Heavy chain: H51, H65 and H99; and

[0427] Light chain: L92, L94, L95 and L96.

[0428] In some embodiments, the isolated anti-C1s antibody of the invention comprises at least one histidine which is a substituted residue at one or more of the following positions and a CDR or FR amino acid position (according to the Kabat numbering system):

[0429] Heavy chain: H51, H65 and H99; and

[0430] Light chain: L92, L94, L95 and L96.

[0431] In some embodiments, the isolated anti-C1s antibody of the invention comprises at least one histidine which is a substituted residue at the following position (according to the Kabat numbering system):

[0432] 1) L92 and L94

[0433] 2) L92 and L95

[0434] 3) L94 and L95

[0435] 4) L92, L94 and L95

[0436] 5) H65 and L92

[0437] 6) H65 and L94

[0438] 7) H65 and L95

[0439] 8) H65, L92 and L94

[0440] 9) H65, L92 and L95

[0441] 10) H65, L94 and L95

[0442] 11) H65, L92, L94 and L95

[0443] 12) H99 and L92

[0444] 13) H99 and L94

[0445] 14) H99 and L95

[0446] 15) H99, L92 and L94

[0447] 16) H99, L92 and L95

[0448] 17) H99, L94 and L95

[0449] 18) H99, L92, L94 and L95

[0450] 19) H65 and H99

[0451] 20) H65, H99 and L92

[0452] 21) H65, H99 and L94

[0453] 22) H65, H99 and L95

[0454] 23) H65, H99, L92 and L94

[0455] 24) H65, H99, L92 and L95

[0456] 25) H65, H99, L94 and L95

[0457] 26) H65, H99, L92, L94 and L95, or

[0458] 27) H27, H99 and L95.

[0459] In any of the above embodiments, the anti-C1s antibody is humanized. In one embodiment, the anti-C1s antibody comprises HVRs as in any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework. In another embodiment, the anti-C1s antibody comprises HVRs as in any of the above embodiments, and further comprises a VH or VL comprising a FR sequence.

[0460] In yet another aspect, the present invention provides antibodies that bind to the same epitope as the anti-C1s antibodies provided herein. For example, in certain embodiments, the present invention provides antibodies that bind to the same epitope as an antibody selected from the group consisting of:

[0461] IPN-M1, IPN-M2, IPN-M3, IPN-M8, IPN-M9, IPN-M10, IPN-M11, IPN-M13, IPN-M14, IPN-M15, IPN-M18, IPN-M23, IPN-M24, IPN-M27, IPN-M28, disclosed in WO2014 / 066744, IPN-M29 and IPN-M33.

[0462] In some embodiments, the isolated anti-C1s antibody of the invention competes for binding to C1s at neutral pH with an antibody selected from the group consisting of:

[0463] IPN-M1, IPN-M2, IPN-M3, IPN-M8, IPN-M9, IPN-M10, IPN-M11, IPN-M13, IPN-M14, IPN-M15, IPN-M18, IPN-M23, IPN-M24, IPN-M27, IPN-M28, disclosed in WO2014 / 066744, IPN-M29 and IPN-M33.

[0464] In one aspect, the present disclosure provides an isolated humanized monoclonal antibody with pH-dependent binding that specifically binds to an epitope within a region encompassing domains IV and V of complement component 1s (Cls). In some cases, the antibody inhibits the binding of Cls to complement component 4 (C4). In some cases, the antibody does not inhibit the protease activity of Cls. In some cases, the epitope bound by the isolated humanized monoclonal antibody of the present disclosure is a conformational epitope.

[0465] In one aspect, the present disclosure provides an isolated antibody with pH-dependent binding that specifically binds to an epitope within a complement Cls protein. In some embodiments, the isolated anti-C1s antibody of the present disclosure binds to an activated Cls protein. In some embodiments, the isolated anti-C1s antibody of the present disclosure binds to an inactive form of Cls. In other examples, the isolated anti-C1s antibody of the present disclosure binds to an activated Cls protein and an inactive form of Cls.

[0466] In one aspect, the present disclosure provides an isolated humanized monoclonal antibody with pH-dependent binding that specifically binds to an epitope within a region covering domains IV and V of C1s. For example, the present disclosure provides an isolated humanized monoclonal antibody that specifically binds to an epitope within amino acids 287-437 of the amino acid sequence set forth in SEQ ID NO: 3. In some cases, the isolated humanized monoclonal antibody specifically binds to an epitope within amino acids 287-437 of the amino acid sequence set forth in SEQ ID NO: 3, and inhibits the binding of C4 to C1s. The present disclosure also provides a method for treating a complement-mediated disease or condition, the method comprising administering to an individual in need thereof an effective amount of an isolated humanized monoclonal antibody that specifically binds to an epitope within amino acids 287-437 of the amino acid sequence set forth in SEQ ID NO: 3, and inhibits the binding of C4 to C1s.

[0467] In one aspect, the present disclosure provides an isolated humanized monoclonal antibody having pH-dependent binding that specifically binds to an epitope comprising aspartic acid at position 372 of the human C1s antigen set forth in SEQ ID NO:3.

[0468] In yet another aspect of the invention, the anti-C1s antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric antibody, a humanized antibody or a human antibody. In one embodiment, the anti-C1s antibody is an antibody fragment, such as a Fv, Fab, Fab', scFv, a diabody or a F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1, IgG2, IgG3 or IgG4 antibody or other antibody classes or isotypes defined herein.

[0469] In yet another aspect, the anti-C1s antibody according to any of the above embodiments may incorporate any of the features, alone or in combination, as described in Sections 1-7 below:

[0470] 1. Antibody affinity

[0471] In certain embodiments, the dissociation constant (Kd or KD) of an antibody provided herein is 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M).

[0472] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, RIA is performed using a Fab form of the target antibody and its antigen. For example, the solution binding affinity of the Fab for the antigen is measured by: in the presence of a series of titrated unlabeled antigens with a minimum concentration of ( 125 I) labeled antigen equilibrated Fab, and then the bound antigen was captured with an anti-Fab antibody coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish the assay conditions, MICROTITER (registered trademark) multiwell plates (ThermoScientific) were coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (about 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125 I]-antigen is mixed with a serial dilution of the target Fab (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The target Fab is then incubated overnight; however, the incubation can be continued for a longer time (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The liquid is then discarded and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20 (registered trademark)) in PBS. When the plate is dry, 150 μL / well scintillant (MICROSCINT-20 TM ; Packard) and in TOPCOUNT TM Plates were counted for 10 minutes on a gamma counter (Packard).Concentrations of each Fab that yielded less than or equal to 20% of maximal binding were selected for competitive binding assays.

[0473] According to another embodiment, KD is measured using BIACORE (registered trademark) surface plasmon resonance assay. For example, the assay using BIACORE (registered trademark) -2000 or BIACORE (registered trademark) -3000 (BIAcore, Inc., Piscataway, NJ) is performed at 25°C using an immobilized antigen CM5 chip with ~10 response units (RU). In one embodiment, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Before injection at a flow rate of 5 μL / min, the antigen is diluted to 5 μg / ml (~0.2 μM) with 10 mM sodium acetate (pH 4.8) to obtain about 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) were injected into a 4% paraformaldehyde-containing 4% paraformaldehyde-containing solution containing 0.05% polysorbate 20 (TWEEN-20 TM ) surfactant in PBS (PBST). The association rate (k on ) and dissociation rate (k off ) was calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE (registered trademark) evaluation software version 3.2). The equilibrium dissociation constant (KD) was calculated as k off / k on See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10 as measured by surface plasmon resonance, 6 M -1 s -1 , the conduction rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing concentrations of antigen, as measured in a spectrometer, such as a stopped-flow spectrophotometer (Aviv Instruments) or a 8000 series SLM-AMINCO with a stirred cuvette. TM Spectrophotometer (ThermoSpectronic).

[0474] In some embodiments, the binding affinity of each histidine substitution variant of the invention at pH 7.4 and pH 5.8 is determined using a Biacore T200 instrument (GE Healthcare) at 37° C. Recombinant protein A / G (Pierce) can be immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes can be prepared in 7(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4), 5(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8), or 5(-) buffer (20 mM ACES, 150 mM NaCl, 3 μM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8). Each antibody can be captured to the sensor surface by protein A / G. The target for antibody capture level is 200 resonance units (RU). Serum-derived human C1s (CompTech) or prepared recombinant C1s can be injected at, for example, 50 or 200 nM and then dissociated. The sensor surface is regenerated in each cycle, for example, using 10 mM glycine-HCl pH 1.5.Binding affinity can be determined by processing the data using, for example, Biacore T200 Evaluation Software Version 2.0 (GE Healthcare) and fitting it to a 1:1 binding model.

[0475] A specific example of the Biacore assay procedure of the present invention is shown below.

[0476] The binding affinity of histidine-substituted variants at pH 7.4 and pH 5.8 was determined using Biacore T200 instrument (GE Healthcare) at 37 ° C. Recombinant protein A / G (Pierce) was fixed on all flow cells of CM4 sensor chips using amine coupling kit (GE Healthcare). Antibodies and analytes were prepared in 7 (+) buffer (20 mMACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4) or 5 (+) buffer (20mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8). Each antibody was captured to the sensor surface by protein A / G. The target of antibody capture level was 200 resonance units (RU). Serum derived human C1s were injected at 12.5, 50 nM (pH 7.4) or 50, 200 nM (pH 5.8) or 200 and 800 nM (pH 5.8) and then dissociated. The sensor surface was regenerated in each cycle using 10 mM glycine-HCl pH 1.5. Binding affinity was determined by processing the data using Biacore T200 Evaluation Software Version 2.0 (GE Healthcare) and fitting it to a 1:1 binding model. Immediately following the dissociation phase at pH 7.4, an additional dissociation phase at pH 5.8 was integrated. The dissociation rate in 5(+) buffer was determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0477] Alternatively, the binding affinity of histidine-substituted variants at pH 7.4 and pH 5.8 was determined using a Biacore T200 instrument (GE Healthcare) at 37°C. Recombinant protein A / G (Pierce) was fixed on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes were prepared in 7 (+) buffer (20 mMACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4) or 5 (+) buffer (20mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8). Each antibody was captured to the sensor surface by protein A / G. The target for antibody capture level was 200 resonance units (RU). Serum-derived human C1s was injected at 50 nM and then dissociated. The sensor surface was regenerated in each cycle using 10 mM glycine-HCl pH 1.5. Binding affinity was determined by processing the data using Biacore T200 Evaluation Software Version 2.0 (GE Healthcare) and fitting it to a 1:1 binding model. Immediately following the dissociation phase at pH 7.4, an additional dissociation phase at pH 5.8 was integrated. The dissociation rate in 5(+) buffer was determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0478] In some embodiments, an additional dissociation phase at pH 5.8 is integrated immediately after the dissociation phase at pH 7.4, if desired. The dissociation rate in 5(+) buffer can be determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0479] 2. Antibody fragments

[0480] In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, Monoclonal Antibody Pharmacology, Vol. 113, Rosenburg and Moore, ed., (Springer-Verlag, NewYork), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments containing salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0481] Diabodies are antibody fragments with two antigen binding sites that can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0482] A single domain antibody is an antibody fragment comprising all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 Bl).

[0483] Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0484] 3. Chimeric and humanized antibodies

[0485] In certain embodiments, the antibodies provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate (e.g., monkey)) and a human constant region. In another example, a chimeric antibody is a "class switching" antibody, in which the class or subclass is changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0486] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or a portion thereof) are derived from non-human antibodies, and FR (or a portion thereof) are derived from human antibody sequences. Humanized antibodies will optionally also include at least a portion of a human constant region. In some embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies derived from HVR residues), for example, to restore or improve antibody specificity or affinity.

[0487] Humanized antibodies and methods for making them are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface remodeling"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and further described in Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" method of FR shuffling).

[0488] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular light or heavy chain variable region subgroup (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0489] 4. Human Antibodies

[0490] In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0491] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to an antigenic challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or it is present outside the chromosome or is randomly incorporated into an animal chromosome. In such transgenic mice, the endogenous immunoglobulin locus is usually inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describe XENOMOUSE TMThe human variable regions of intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0492] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. (See, e.g., Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Technology and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991). Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described in, e.g., U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0493] It is also possible to produce human antibodies by isolating the Fv clone variable domain sequence selected from the human phage display library. Such variable domain sequences can then be combined with desired human constant domains. The technology of selecting human antibodies from the antibody library is as described below.

[0494] 5. Library-derived antibodies

[0495] The antibodies of the invention can be isolated by screening combinatorial libraries to obtain antibodies with the desired activity.For example, various methods are known in the art for generating phage display libraries and screening such libraries to obtain antibodies with the desired binding properties. Such methods are reviewed in, e.g., Hoogenboom et al., Methods in Mol. Biol. 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001), and further described in, e.g., McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Mol. Biol. 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093. (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0496] In some phage display methods, VH and VL gene libraries are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened to obtain antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages usually display antibody fragments in the form of single-chain Fv (scFv) fragments or Fab fragments. Libraries from immune sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, an initial library (e.g., from humans) can be cloned to provide a single source of antibodies to a variety of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, the initial library can also be prepared synthetically by cloning non-rearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and complete rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0497] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0498] 6. Multispecific Antibodies

[0499] In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for C1s, and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of C1s. Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing C1s. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0500] Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs of different specificities (see, Milstein and Cuello, Nature, 305: 537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to prepare bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368-5476 (1994)). (1994)); and preparing trispecific antibodies as described by Tutt et al. J. Immunol. 147: 60 (1991).

[0501] Engineered antibodies with three or more functional antigen binding sites, including "octopus antibodies," are also included herein (see, e.g., US 2006 / 0025576A1).

[0502] The antibodies or fragments herein also include "dual-acting Fabs" or "DAFs," which comprise an antigen binding site that binds C1s as well as another, different antigen (eg, see, US 2008 / 0069820).

[0503] 7. Antibody variants

[0504] In certain embodiments, it is contemplated that the amino acid sequence variants of the antibodies provided herein. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, the deletion and / or insertion and / or replacement of residues in the antibody amino acid sequence. Any combination of deletion, insertion and replacement can be performed to reach the final construct, provided that the final construct has a desired feature, such as antigen binding.

[0505] a) Substitution, insertion and deletion variants

[0506] In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutagenesis include HVR and FR. Conservative substitutions are shown under the heading "preferred substitutions" in Table 1. More substantial changes are provided under the heading "exemplary substitutions" in Table 1, and are further described below with reference to amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0507] [Table 1]

[0508]

[0509] Amino acids can be grouped according to common side chain properties:

[0510] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0511] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0512] (3) Acidic: Asp, Glu;

[0513] (4) Basic: His, Lys, Arg;

[0514] (5) Residues that affect chain orientation: Gly, Pro;

[0515] (6) Aromatic: Trp, Tyr, Phe.

[0516] Non-conservative substitutions will necessitate exchanging a member of one of these classes for another class.

[0517] One type of substitutional variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Typically, the resulting variant selected for further study will have modifications (e.g., improvements) and / or will substantially retain certain biological properties of the parent antibody in terms of certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody. Exemplary substitutional variants are affinity-matured antibodies that can be conveniently produced, for example, using affinity maturation techniques based on phage display, such as those described herein. In short, one or more HVR residues are mutated and the variant antibody is displayed on phage and screened for specific biological activity (e.g., binding affinity).

[0518] Changes (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such changes can be made in HVR "hot spots", i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or residues that contact antigens, and the resulting variant VH or VL binding affinity is tested. Affinity maturation by construction and reselection from a secondary library is described, for example, in Hoogenboom et al. Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variant with a desired affinity. Another method for introducing diversity involves an HVR-directed approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

[0519] In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity may be made in HVR. For example, such changes may be outside the antigen contact residues in HVR. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two or three amino acid substitutions.

[0520] A kind of available method for identifying the residue or region of the antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis", as described in Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or a group of target residues (e.g., charged residues, such as arg, asp, his, lys and glu) are identified and replaced by neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further replacements can be introduced at amino acid positions that show functional sensitivity to the initial replacement. Alternatively, or additionally, the crystal structure of the antigen-antibody complex can be analyzed to identify the contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as replacement candidates. Variants can be screened to determine whether they contain desired properties.

[0521] Amino acid sequence insertions include amino and / or carboxyl terminal fusions of polypeptides ranging in length from one residue to one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of enzymes (e.g., for ADEPT) or polypeptides (which increase the plasma half-life of antibodies) to the N-terminus or C-terminus of the antibody.

[0522] b) Glycosylation variants

[0523] In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent to which the antibodies are glycosylated. Addition or deletion of glycosylation sites to antibodies can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0524] In the case where the antibody comprises the Fc district, the carbohydrate attached thereto can be changed. The natural antibody produced by mammalian cells generally comprises the biantennary oligosaccharide of branching, which is generally attached to the Asn297 of the CH2 domain of the Fc district by an N bond. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose attached to GlcNAc in the " stem " of biantennary oligosaccharide structure. In some embodiments, the oligosaccharide in the antibody of the present invention can be modified to produce antibody variants with some improved properties.

[0525] In one embodiment, an antibody variant having a carbohydrate structure is provided, which lacks fucose connected (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 sugar chain (relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) connected to Asn 297), as measured by MALDI-TOF mass spectrometry, for example as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about position 297 in the Fc region (EU numbering of residues in the Fc region); however, due to minor sequence variations in antibodies, Asn297 may also be located at about + / -3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, U.S. Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene FUT8 knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0526] Antibody variants further provide bisected oligosaccharides, for example, wherein the biantennary oligosaccharides connected to the antibody Fc region are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide connected to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0527] c) Fc region variants

[0528] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby producing an Fc region variant. An Fc region variant can be included in a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., replacement) at one or more amino acid positions.

[0529] In certain embodiments, the present invention contemplates antibody variants with some but not all effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important but certain effector functions (e.g., complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm that CDC and / or ADCC activity is reduced / depleted. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays can be used (see, e.g., ACT1 by flow cytometry). TMNon-radioactive cytotoxicity assays (CellTechnology, Inc. Mountain View, CA; and CytoTox 96 (registered trademark) non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the target molecule can be assessed in vivo, for example in an animal model, such as that disclosed by Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO 2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determination can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0530] Antibodies with reduced effector function include antibodies with one or more Fc region residues 238, 265, 269, 270, 297, 327, and 329 substitutions (U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more positions of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with residues 265 and 297 substituted with alanine (U.S. Pat. No. 7,332,581).

[0531] Certain antibody variants with increased or decreased binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0532] In certain embodiments, the antibody variants comprise an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (EU numbering of residues) of the Fc region.

[0533] In some embodiments, alterations are made in the Fc region that result in altered (i.e., increased or decreased) Clq binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0534] Antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US 2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions therein, wherein the substitutions increase binding of the Fc region to FcRn. Such Fc variants include variants having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424 or 434, e.g., substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

[0535] For other examples of Fc region variants, see Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351.

[0536] d) Cysteine ​​engineered antibody variants

[0537] In certain embodiments, it may be desirable to construct cysteine ​​engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are replaced by cysteine ​​residues. In a specific embodiment, the replaced residues appear at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thus located at accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be replaced by cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine ​​engineered antibodies can be produced as described in, for example, U.S. Patent No. 7,521,541.

[0538] e) Antibody derivatives

[0539] In certain embodiments, the antibodies provided herein may be further modified to include additional non-proteinaceous moieties known in the art and readily available. Suitable portions for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylene polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Due to its stability in water, polyethylene glycol propionaldehyde may have advantages in production. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be identical or different molecules. Generally, the amount and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used therapeutically under defined conditions, and the like.

[0540] In another embodiment, a conjugate of an antibody and a non-protein moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, and includes but is not limited to wavelengths that do not harm normal cells but heat the non-protein moiety to a temperature close to that at which cells of the antibody-non-protein moiety are killed.

[0541] B. Recombinant Methods and Compositions

[0542] Recombinant methods and compositions can be used to produce antibodies, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-C1s antibody described herein is provided. Such nucleic acid can encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light chain and / or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In another embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, the host cell comprises the following (e.g., has been transformed by the following): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., a Y0, NS0, Sp2 / 0 cell). In one embodiment, a method of preparing an anti-C1s antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0543] For recombinant production of anti-C1s antibodies, nucleic acids encoding the antibodies (e.g., as described above) are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibodies).

[0544] Suitable host cells for cloning or expressing antibody encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in Escherichia coli). After expression, antibodies can be separated from bacterial cell suspensions in soluble fractions and can be further purified.

[0545] In addition to prokaryotes, eukaryotic microorganisms (e.g., filamentous fungi or yeast) are suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized" to produce antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004), and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0546] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0547] Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TM technology).

[0548] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Examples of other useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells, as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT060562); TRI cells, e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68. (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0549] Antibodies with pH-dependent characteristics can be obtained by using screening methods and / or mutagenesis methods, for example, as described in WO2009 / 125825. Screening methods can include any process of identifying antibodies with pH-dependent binding characteristics in an antibody group specific for a particular antigen. In certain embodiments, screening methods can include measuring one or more binding parameters (e.g., KD or kd) of a single antibody in an initial antibody group at acidic pH and neutral pH. The binding parameters of antibodies can be measured using, for example, surface plasmon resonance or any other analytical method that allows quantitative or qualitative assessment of the binding properties of antibodies to specific antigens. In certain embodiments, screening methods can include identifying antibodies that are binding antigens with an acidic KD / neutral KD ratio of 2 or greater. Alternatively, screening methods can include identifying antibodies that are binding antigens with an acidic kd / neutral kd ratio of 2 or greater.

[0550] In another embodiment, the mutagenesis method may include the incorporation of a deletion, substitution or addition of an amino acid into the heavy chain and / or light chain of the antibody to enhance the pH-dependent binding of the antibody to the antigen. In certain embodiments, mutagenesis may be performed within one or more variable domains of the antibody, for example, within one or more HVRs (e.g., CDRs). For example, mutagenesis may include replacing an amino acid within one or more HVRs (e.g., CDRs) of the antibody with another amino acid. In certain embodiments, mutagenesis may include replacing one or more amino acids in at least one HVR (e.g., CDR) of the antibody with histidine. In certain embodiments, "enhanced pH-dependent binding" refers to a mutant form of the antibody that exhibits a greater acidic KD / neutral KD ratio, or a greater acidic kd / neutral kd ratio, compared to the original "parent" form of the antibody before mutagenesis. In certain embodiments, the acidic KD / neutral KD ratio of the mutant form of the antibody is 2 or greater. Alternatively, the acidic kd / neutral kd ratio of the mutant form of the antibody is 2 or greater.

[0551] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) using a bifunctional or derivatizing agent (e.g., maleimidobenzoylsulfosuccinimide ester (conjugated through cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR, wherein R and R1 are different alkyl groups). .

[0552] Animals (usually non-human mammals) are immunized against antigens, immunogenic conjugates or derivatives by, for example, combining 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animal is boosted with 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, the animal is bled and the antibody titer of the serum is determined. The animal is boosted until the titer plateau is reached. Preferably, the animal is boosted with conjugates of the same antigen conjugated to different proteins and / or by different cross-linking agents. Conjugates can also be prepared in recombinant cell cultures as protein fusions. In addition, aggregating agents (e.g., alum) are also suitable for enhancing immune responses.

[0553] A monoclonal antibody is obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0554] For example, monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al., Nature 256(5517):495-497 (1975). In the hybridoma method, mice or other suitable host animals, such as hamsters, are immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro.

[0555] Immunizing agents will generally include antigenic proteins or fusion variants thereof. Typically, if cells of human origin are required, peripheral blood lymphocytes (PBL) are used, or if cells of non-human mammalian origin are required, spleen cells or lymph node cells are used. Lymphocytes are then fused to immortalized cell lines using a suitable fusing agent (e.g., polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0556] Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells from rodent, cattle and people.Usually, rat or mouse myeloma cell lines are used.The hybridoma cells prepared in this way are inoculated and grown in a suitable culture medium, which preferably contains one or more substances that suppress the growth or survival of the unfused parental myeloma cells.For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium of the hybridoma usually includes hypoxanthine, aminopterin and thymidine (HAT culture medium), which are substances that stop HGPRT from lacking cell growth.

[0557] Preferred immortalized myeloma cells are those that effectively fuse, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to culture media (e.g., HAT culture media). Among them, preferred are mouse myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors obtained from the Salk Institute Cell Distribution Center in San Diego, California, USA, and cell lines obtained from SP-2 cells (and derivatives thereof, e.g., X63-Ag8-653) obtained from the American Type Culture Collection, Manassas, Virginia, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor et al., J. Immunol. 133(6):3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Technology and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

[0558] The culture medium in which the hybridoma cells are grown is measured for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. For example, binding affinity can be determined by Scatchard analysis of Munson, Anal. Biochem. 107 (1): 220-239 (1980).

[0559] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity and / or activity, the clones can be divided into subclones by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 culture media. In addition, hybridoma cells can be grown as tumors in mammals.

[0560] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0561] C. Determination

[0562] The anti-C1s antibodies provided herein can be identified, screened or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0563] 1. Binding assays and other assays

[0564] In one aspect, for example, the antigen binding activity of the antibodies of the present invention is detected by known methods (eg, ELISA, Western blotting, etc.).

[0565] In another aspect, competition assays can be used to identify antibodies that compete with any of the anti-C1s antibodies described herein for binding to C1s. In certain embodiments, when such competing antibodies are present in excess, they block (e.g., reduce) binding of a reference antibody to C1s by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as any of the anti-C1s antibodies described herein. Detailed exemplary methods for mapping epitopes to which antibodies bind are provided in Morris (1996) "Epitope Mapping Protocols", Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ). In certain embodiments, such competition assays can be conducted under neutral pH conditions.

[0566] In an exemplary competition assay, immobilized C1s is incubated in a solution comprising a first labeled antibody (e.g., one of those described herein) that binds to C1s and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to C1s. The second antibody can be present in the hybridoma supernatant. As a control, immobilized C1s is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to C1s, excess unbound antibody is removed and the amount of label associated with immobilized C1s is measured. If the amount of label associated with immobilized C1s in the test sample is substantially reduced relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to C1s. See Harlow and Lane (1988) Antibodies: A Laboratory Manual Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0567] On the other hand, antibodies that bind to the same epitope as the anti-C1s antibodies provided herein or compete with the anti-C1s antibodies provided herein for binding to C1s can be identified using a sandwich assay. A sandwich assay involves the use of two antibodies, each of which is capable of binding to a different immunogenic portion or epitope of the protein to be detected. In a sandwich assay, a test sample analyte binds to a first antibody immobilized on a solid support, and then a second antibody binds to the analyte, thereby forming an insoluble three-part complex. See David & Greene, U.S. Patent No. 4,376,110. The second antibody itself can be labeled with a detectable moiety (direct sandwich assay) or can be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme. An antibody that binds to C1s simultaneously with the anti-C1s antibodies provided herein can be identified as an antibody that binds to a different epitope than the anti-C1s antibody. Therefore, an antibody that does not bind to C1s simultaneously with the anti-C1s antibody provided herein can be identified as an antibody that binds to the same epitope as the anti-C1s antibody or that competes with the anti-C1s antibody for binding to C1s.

[0568] 2. Activity Assay

[0569] In one aspect, an assay is provided for identifying an anti-C1s antibody having biological activity. The biological activity may include blocking activation of the classical pathway and the production of cleavage products (C2a, C2b, C3a, C3b, C4a, C4b, C5a and C5b) resulting from activation of the pathway. Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0570] In certain embodiments, such biological activities of the antibodies of the present invention are detected. In some embodiments, the ability of the antibodies of the present invention to inhibit complement-mediated hemolysis of chicken red blood cells (cRBC) sensitized with antibodies to cRBC antigens can be evaluated. Using human serum as a source of complement proteins, the activity of the antibodies of the present invention can be determined by spectrophotometrically measuring the amount of released hemoglobin. In some embodiments, the ability of the antibodies of the present invention to inhibit the cleavage of activated C1s-mediated purified C4 but not C2 can be evaluated. The activity of the antibody is determined by measuring the amount of cleaved C4 or C2 by gel electrophoresis or western blotting. Compared with its native uncleaved form, cleaved C4 or C2 can be detected by its smaller molecular weight.

[0571] 3. Mouse PK studies to evaluate antigen (C1s) elimination

[0572] In certain embodiments, acceleration of antigen (eg, human C1s (also referred to as hC1s)) elimination by antibodies of the invention can be assessed in vivo (eg, in mice) as follows.

[0573] Measurement of C1s concentration in mouse plasma by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC / ESI-MS / MS)

[0574] The concentration of hC1s (or anti-C1s antibody) in mouse plasma can be measured by LC / ESI-MS / MS. Calibration standards are prepared by mixing and diluting hC1s (or anti-C1s antibody) in mouse plasma in specified amounts. Calibration standards and plasma samples are mixed with, for example, urea, dithiothreitol, and lysozyme (hen egg white) in ammonium bicarbonate and incubated. Then, iodoacetamide is added and incubated in the dark. Next, trypsin in ammonium bicarbonate is added and incubated. Finally, trifluoroacetic acid is added to inactivate any residual trypsin. The sample is analyzed by LC / ESI-MS / MS. Human C1s-specific peptides (e.g., LLEVPEGR) are monitored by selected reaction monitoring (SRM). For human C1, the SRM transition may be [M+2H]2+ (m / z456.8 to y6 ion (m / z 686.4)). A calibration curve can be constructed by weighted (1 / x2) linear regression using peak areas plotted against concentration. Concentrations in mouse plasma were calculated from the calibration curve.

[0575] Pharmacokinetic evaluation of total hC1s after administration of anti-C1s antibody in mice

[0576] The in vivo pharmacokinetics of hCls, hClq or anti-C1s antibodies can be evaluated after administering the antigen alone or in combination with the anti-C1s antibody to mice. A solution / mixture containing hCls (etc.) is intravenously injected into mice. Immediately after administration of the antigen solution, the anti-C1s antibody solution is administered to the same individual in the same manner. The dose setting can be appropriately designed to allow almost all hCls to be in a bound form in the circulation. Blood is collected over time (e.g., 5, 30 minutes, 2, 7 hours, 3, 7, 14, 21 and 28 days after injection). The blood is immediately centrifuged to separate the plasma sample. The plasma concentration of hCls (etc.) is measured at each sampling point by LC / ESI-MS / MS. The PK parameters of hCls (etc.) are estimated by non-compartmental analysis. For example, the hCls CL (clearance rate) ratio of the anti-C1s antibody is calculated. If the ratio is higher, it means that hCls can be eliminated faster.

[0577] D. Immunoconjugates

[0578] The invention also provides immunoconjugates comprising an anti-C1s antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes.

[0579] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0580] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcosine, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, Curculin toxin, crotonin, saponin inhibitor, gelonin, mitogenins, restrictocins, phenomycins, enomycins, and trichothecenes.

[0581] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used to produce radioconjugates. Examples include 211 At 131 I. 125 I. 90 Y. 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P. 212 When the radioconjugate is used for detection, it may contain a radioactive atom such as Tc-99m or 123 I, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[0582] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipate), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bisazido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisazido derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" that promotes the release of cytotoxic drugs in cells. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020).

[0583] The immunoconjugates or ADCs herein specifically contemplate, but are not limited to, such conjugates prepared with cross-linking agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfoEMCS, sulfoGMBS, sulfoKMUS, sulfoMBS, sulfoSIAB, sulfoSMCC, sulfoSMPB, and SVSB (succinimidyl-(4-vinyl sulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0584] E. Methods and compositions for diagnosis and detection

[0585] In certain embodiments, any of the anti-C1s antibodies provided herein can be used to detect the presence of C1s in a biological sample. As used herein, the term "detection" includes quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues, such as serum, whole blood, plasma, biopsy samples, tissue samples, cell suspensions, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, ascites, milk, colostrum, mammary secretions, lymph, urine, sweat, tears, gastric fluid, synovial fluid, peritoneal fluid, lens fluid or mucus.

[0586] In one embodiment, an anti-C1s antibody for use in a diagnostic or detection method is provided. In another aspect, a method for detecting the presence of C1s in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-C1s antibody described herein under conditions that allow the anti-C1s antibody to bind to C1s, and detecting whether a complex is formed between the anti-C1s antibody and C1s. This method can be an in vitro or in vivo method. In one embodiment, the anti-C1s antibody is used to select subjects suitable for anti-C1s antibody therapy, for example, where C1s is a biomarker for selecting patients.

[0587] Exemplary disorders that can be diagnosed using the antibodies of the invention include, but are not limited to, age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, allergic reactions, argyrophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behcet's disease, British type amyloid angiopathy, bullous pemphigoid, Buerger's disease, Clq nephropathy, cancer, catastrophic antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Crohn's disease, cryoglobulinemic vasculitis, dementia pugilistica, dementia with Lewy bodies, Bodies (DLB), diffuse neurofibrillary tangles with calcifications, discoid lupus erythematosus, Down syndrome, focal segmental glomerulosclerosis, formal thought disorder, frontotemporal dementia (FTD), Parkinson's disease associated with chromosome 17, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker disease, Guillain-Barre syndrome, Hall-Straussler disease, hemolytic uremic syndrome, hereditary angioedema, hypophosphatemia, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infectious diseases (e.g., caused by bacteria (e.g., Neisseria meningitidis or Streptococcus), viruses (e.g.,diseases caused by human immunodeficiency virus (HIV) or other infectious agents), inflammatory diseases, ischemia / reperfusion injury, mild cognitive impairment, immune thrombocytopenic purpura (ITP), molybdenum cofactor deficiency type A deficiency)(MoCD), membranoproliferative glomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis (MPGN) II (dense deposit disease), membranous nephritis, multi-infarct dementia, lupus (e.g., systemic lupus erythematosus (SLE)), glomerulonephritis, Kawasaki disease, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, myasthenia gravis, myocardial infarction, myotonic dystrophy, neuromyelitis optica, Niemann-Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Parkinson disease, Parkinson disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, postencephalitic parkinsonism, polymyositis, prion protein cerebral amyloid angiopathy angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga toxin-producing Escherichia coli (STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, Tangle only dementia, transplant rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegner's granulomatosis, sickle cell disease, cryoglobulinemia, mixed cryoglobulinemia, essential mixed cryoglobulinemia, mixed cryoglobulinemia type II, mixed cryoglobulinemia type III, nephritis, drug-induced thrombocytopenia, lupus nephritis, bullous pemphigoid, epidermolysis bullosa acquisita, delayed hemolytic transfusion reaction, hypocomplementemic urticarial vasculitis syndrome, pseudophakic bullous keratopathy, and platelet refractoriness.

[0588] In certain embodiments, labeled anti-C1s antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), and indirectly detectable moieties such as enzymes or ligands, e.g., via an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, radioisotopes. 32 P. 14 C. 125 I. 3 H and 131I, fluorophores (e.g., rare earth chelates or fluorescein and its derivatives), rhodamine and its derivatives, dansyl, umbelliferone, luciferase (e.g., firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456)), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-glucosamine, glucoamylase, lysozyme, sugar oxidases (e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), those using hydrogen peroxide to oxidize dye precursors (e.g., HRP, lactoperoxidase or microperoxidase, biotin / avidin, spin labels, phage labels, stable free radicals, etc.) conjugated to enzymes.

[0589] F. Pharmaceutical Preparations

[0590] Pharmaceutical preparations of antigen binding molecules as described herein are prepared by mixing such molecules with the desired purity with one or more optional pharmaceutically acceptable carriers (Remington Pharmaceutical Science 16th Edition, Osol, A. Editor (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to the recipient at the dosage and concentration employed, and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and metacresol); low molecular weight (less than about 10 Residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose or sorbitol; counterions to form salts, such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is used in combination with one or more additional glycosaminoglycanases, such as chondroitinases.

[0591] Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter including a histidine-acetate buffer.

[0592] The formulations herein may also contain more than one active ingredient, preferably active ingredients having complementary activities and not adversely affecting each other, as necessary for the specific indication to be treated. For example, it may be desirable to further provide a formulation for combined therapy. Such active ingredients are appropriately combined in an amount effective for the intended purpose.

[0593] The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Science 16th edition, Osol, A. ed. (1980).

[0594] Sustained release preparations may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the molecule, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

[0595] Preparations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

[0596] G. Methods of treatment and compositions

[0597] Any of the antigen binding molecules provided herein can be used in therapeutic methods.

[0598] On the one hand, antigen binding molecules for use as medicines are provided. In another aspect, antigen binding molecules for use in the treatment of complement-mediated diseases or conditions are provided. In certain embodiments, antigen binding molecules for use in methods of treatment are provided. In certain embodiments, the invention provides antigen binding molecules for use in methods of treating individuals suffering from complement-mediated diseases or conditions, the method comprising administering an effective amount of antigen binding molecules to the individual. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual.

[0599] In a further embodiment, the present invention provides an antigen binding molecule for treating a complement-mediated disease or condition. In a further embodiment, the antigen binding molecule of the present invention can be used to enhance the clearance of C1s from plasma. In a further embodiment, the antigen binding molecule of the present invention can be used to enhance the clearance of C1q, C1r and C1s complexes from plasma. In some embodiments, the antigen binding molecule of the present invention can be used to inhibit the cleavage of complement component C4, wherein the molecule does not inhibit the cleavage of complement component C2. In some cases, the molecule inhibits components of the classical complement pathway; in some cases, the classical complement pathway component is Cls. In certain embodiments, the present invention provides an antigen binding molecule in a method for treating a complement-mediated disease or condition. In certain embodiments, the present invention provides an antigen binding molecule in a method for enhancing the clearance of C1s from plasma. In certain embodiments, the present invention provides an antigen binding molecule in a method for enhancing the clearance of C1q, C1r and C1s complexes from plasma. In certain embodiments, the present invention provides an antigen binding molecule in a method for inhibiting the cleavage of complement component C4, wherein the molecule does not inhibit the cleavage of complement component C2. In certain embodiments, the invention provides an antigen binding molecule for use in a method of inhibiting a component of the classical complement pathway; in some cases, the classical complement pathway component is Cls. An "individual" according to any of the above embodiments is preferably a human.

[0600] On the one hand, the present disclosure provides a method for regulating complement activation. In some embodiments, the method inhibits complement activation, for example to reduce the production of C4b2a. In some embodiments, the present disclosure provides a method for regulating complement activation in an individual suffering from a complement-mediated disease or condition, the method comprising administering an antigen binding molecule of the present disclosure or a pharmaceutical composition of the present disclosure to the individual, wherein the pharmaceutical composition comprises an antigen binding molecule of the present disclosure. In some embodiments, this method inhibits complement activation. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. It can be administered by any route known to those skilled in the art, including those disclosed herein. In some embodiments, administration is intravenous or subcutaneous administration. In some embodiments, administration is intrathecal administration.

[0601] A complement-mediated disease or disorder is a disorder characterized by an abnormal amount of complement Cls or an abnormal level of complement Cls proteolytic activity in the cells, tissues or fluids of an individual.

[0602] In some cases, the disease or condition of complement mediation is characterized by the presence of an elevated (higher than normal) amount of Cls or an elevated level of complement Cls activity in a cell, tissue or fluid. For example, in some cases, the disease or condition of complement mediation is characterized by an elevated amount and / or elevated Cls activity in brain tissue and / or cerebrospinal fluid. The Cls amount "higher than normal" in a cell, tissue or fluid indicates that the Cls amount in the cell, tissue or fluid is higher than the normal control level, for example, higher than the normal control level of an individual or individual population of the same age group. The "higher than normal" level of Cls activity in a cell, tissue or fluid indicates that the proteolytic cleavage of Cls influence in a cell, tissue or fluid is higher than the normal control level, for example, higher than the normal control level of an individual or individual population of the same age group. In some cases, an individual suffering from a disease or condition of complement mediation shows one or more additional symptoms of this disease or condition.

[0603] In other cases, the complement-mediated disease or condition is characterized by the presence of a lower than normal amount of Cls or a lower level of complement Cls activity in a cell, tissue or fluid. For example, in some cases, the complement-mediated disease or condition is characterized by the presence of a lower amount and / or lower activity of Cls in brain tissue and / or cerebrospinal fluid. The amount of Cls "lower than normal" in a cell, tissue or fluid indicates that the amount of Cls in the cell, tissue or fluid is lower than the normal control level, for example, lower than the normal control level for an individual or individual population of the same age group. The "lower than normal" level of Cls activity in a cell, tissue or fluid indicates that the proteolytic cleavage affected by Cls in the cell, tissue or fluid is lower than the normal control level, for example, lower than the normal control level for an individual or individual population of the same age group. In some cases, an individual suffering from a complement-mediated disease or condition exhibits one or more additional symptoms of this disease or condition.

[0604] A complement-mediated disease or condition is a disease or condition in which the amount or activity of complement C1s causes the disease or condition to occur in an individual. In some embodiments, the complement-mediated disease or condition is selected from the group consisting of autoimmune diseases, cancer, blood diseases, infectious diseases, inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, neurodegenerative disorders, eye diseases, kidney diseases, transplant rejection, vascular diseases, and vasculitis diseases. In some embodiments, the complement-mediated disease or condition is an autoimmune disease. In some embodiments, the complement-mediated disease or condition is cancer. In some embodiments, the complement-mediated disease or condition is an infectious disease. In some embodiments, the complement-mediated disease or condition is an inflammatory disease. In some embodiments, the complement-mediated disease or condition is a blood disease. In some embodiments, the complement-mediated disease or condition is ischemia-reperfusion injury. In some embodiments, the complement-mediated disease or condition is an eye disease. In some embodiments, the complement-mediated disease or condition is kidney disease. In some embodiments, the complement-mediated disease or condition is transplant rejection. In some embodiments, the complement-mediated disease or condition is antibody-mediated transplant rejection. In some embodiments, the complement-mediated disease or condition is a vascular disease. In some embodiments, the complement-mediated disease or condition is a vasculitis condition. In some embodiments, the complement-mediated disease or condition is a neurodegenerative disease or condition. In some embodiments, the complement-mediated disease is a neurodegenerative disease. In some embodiments, the complement-mediated disease is a neurodegenerative disease. In some embodiments, the complement-mediated disease or condition is a neurodegenerative condition. In some embodiments, the complement-mediated disease or condition is a tauopathy.

[0605] Examples of complement-mediated diseases or disorders include, but are not limited to, age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, allergic reactions, argyrophilic dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behcet's disease, British amyloid angiopathy, bullous pemphigoid, Buerger's disease, C1q nephropathy, cancer, catastrophic antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt-Jakob disease, ob disease, Crohn's disease, cryoglobulinemia, dementia pugilistica, dementia with Lewy bodies (DLB), diffuse neurofibrillary tangles with calcifications, discoid lupus erythematosus, Down syndrome, focal segmental glomerulosclerosis, formal thought disorder, frontotemporal dementia (FTD), Parkinson's disease associated with chromosome 17, frontotemporal lobar dementia, Gerstmann-Straussler-Scheinker disease, Guillain-Barre syndrome, Hartmann-Straussler disease, hemolytic uremic syndrome, hereditary angioedema, hypophosphatemia, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infectious diseases 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 type A (MoCD), membranoproliferative glomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis (MPGN) II (dense deposit disease), membranous nephritis, multi-infarct dementia, lupus (e.g., systemic lupus erythematosus (SLE)), glomerulonephritis, Kawasaki disease, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, Myasthenia gravis, myocardial infarction, myotonic dystrophy, neuromyelitis optica, Niemann-Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Parkinson's disease, Parkinson's disease with dementia, paroxysmal nocturnal hemoglobinuria, pemphigus vulgaris, Pick's disease, postencephalitic Parkinson's disease, polymyositis, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, psoriasis, sepsis, Shiga toxin-producing Escherichia coli (STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, tangled dementia, transplant rejection, vasculitis (e.g.,ANCA-associated vasculitis), Wegner's granulomatosis, sickle cell disease, cryoglobulinemia, mixed cryoglobulinemia, essential mixed cryoglobulinemia, mixed cryoglobulinemia type II, mixed cryoglobulinemia type III, nephritis, drug-induced thrombocytopenia, lupus nephritis, bullous pemphigoid, epidermolysis bullosa acquisita, delayed hemolytic transfusion reaction, hypocomplementemic urticarial vasculitis syndrome, pseudophakic bullous keratopathy, and platelet transfusion failure.

[0606] Alzheimer's disease and certain forms of frontotemporal dementia (Pick's disease, sporadic frontotemporal dementia and Parkinson's disease frontotemporal dementia associated with chromosome 17) are the most common forms of tauopathy. Accordingly, the present invention relates to any method as described above, wherein the tauopathy is Alzheimer's disease, Pick's disease, sporadic frontotemporal dementia and Parkinson's disease frontotemporal dementia associated with chromosome 17. Other tauopathies include, but are not limited to, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) (CBD) and subacute sclerosing panencephalitis.

[0607] Neurodegenerative tauopathies include Alzheimer's disease, amyotrophic lateral sclerosis / Parkinson's disease-dementia complex, argyrophilic dementia, British amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangles with calcifications, Down syndrome, frontotemporal dementia, frontotemporal dementia with Parkinson's disease associated with chromosome 17, and frontotemporal lobar degeneration. degeneration), Gerstmann-Straussler-Scheinker disease, Haas-Straussler disease, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guamanian motor neuron disease with neurofibrillary tangles, Pick's disease, postencephalitic Parkinsonism, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangled dementia, multi-infarct dementia, ischemic stroke, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), and stroke.

[0608] The present disclosure also provides methods for treating synucleinopathy (e.g., Parkinson's disease (PD); dementia with Lewy bodies (DLB); multiple system atrophy (MSA), etc. For example, PD with dementia (PDD) can be treated with the methods of the present disclosure.

[0609] In some embodiments, the complement-mediated disease or condition comprises Alzheimer's disease. In some embodiments, the complement-mediated disease or condition comprises Parkinson's disease. In some embodiments, the complement-mediated disease or condition comprises transplant rejection. In some embodiments, the complement-mediated disease or condition is antibody-mediated transplant rejection.

[0610] In some embodiments, the antigen binding molecules of the present disclosure prevent or delay the onset of at least one symptom of a complement-mediated disease or condition in an individual. In some embodiments, the antigen binding molecules of the present disclosure reduce or eliminate at least one symptom of a complement-mediated disease or condition in an individual. Examples of symptoms include, but are not limited to, symptoms associated with the following items: autoimmune diseases, cancer, blood diseases, infectious diseases, inflammatory diseases, ischemia-reperfusion injury, neurodegenerative diseases, neurodegenerative conditions, nephropathy, transplant rejection, eye diseases, vascular diseases, or vasculitis. Symptoms can be nervous system symptoms, such as impaired cognitive function, memory impairment, loss of motor function, etc. Symptoms can also be the activity of Cls proteins in individual cells, tissues, or fluids. Symptoms can also be the degree of complement activation in individual cells, tissues, or fluids.

[0611] In some embodiments, the antigen binding molecules of the present disclosure are administered to individuals to regulate complement activation in individual cells, tissues or fluids. In some embodiments, the antigen binding molecules of the present disclosure are administered to individuals to inhibit complement activation in individual cells, tissues or fluids. For example, in some embodiments, the antigen binding molecules of the present disclosure, when administered to individuals suffering from complement-mediated diseases or disorders in one or more doses as a monotherapy or conjoint therapy, inhibit complement activation in individuals by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%.

[0612] In some embodiments, the antigen binding molecules of the present disclosure reduce C3 deposition onto erythrocytes; for example, in some embodiments, the antigen binding molecules of the present disclosure reduce C3b, iC3b, etc. deposition onto RBCs. In some embodiments, the antigen binding molecules of the present disclosure inhibit complement-mediated erythrocyte lysis.

[0613] In some embodiments, the antigen binding molecules of the present disclosure reduce the deposition of C3 onto platelets; for example, in some embodiments, the antigen binding molecules of the present disclosure reduce the deposition of C3b, iC3b, etc. onto platelets.

[0614] In some embodiments, administration of an antigen binding molecule of the present disclosure results in a result selected from the group consisting of: (a) reduced complement activation; (b) improved cognitive function; (c) reduced neuronal loss; (d) reduced levels of phospho-Tau in neurons; (e) reduced glial activation; (f) reduced lymphocyte infiltration; (g) reduced macrophage infiltration; (h) reduced antibody deposition, (i) reduced glial cell loss; (j) reduced oligodendrocyte loss; (k) reduced dendritic cell infiltration; (i) reduced neutrophil infiltration; (m) reduced erythrocyte lysis. ; (n) decreased erythrocyte phagocytosis; (o) decreased platelet phagocytosis; (p) decreased platelet lysis; (q) improved graft survival; (r) decreased macrophage-mediated phagocytosis; (s) improved visual acuity; (t) improved motor control; (u) improved thrombosis; (v) improved coagulation; (w) improved renal function; (x) decreased antibody-mediated complement activation; (y) decreased autoantibody-mediated complement activation; (z) improved anemia; (aa) decreased demyelination; (ab) decreased eosinophilia; (ac) decreased C3 deposition on erythrocytes (e.g., (a) decreased erythrocyte destruction due to transfusion reactions; (b) decreased erythrocyte lysis due to alloantibodies; (c) decreased platelet lysis due to transfusion reactions; (d) decreased platelet lysis due to alloantibodies; (e) decreased anaphylatoxin production; (e) decreased autoantibody-mediated blister formation; (f) decreased autoantibody-induced pruritus; (ah) decreased autoantibody-induced erythema; (ai) decreased autoantibody-mediated skin erosions; (aj) decreased erythrocyte destruction due to transfusion reactions; (ak) decreased erythrocyte lysis due to alloantibodies; (al) decreased hemolysis due to transfusion reactions; (am) decreased alloantibody-mediated platelet lysis; (an) decreased platelet lysis due to transfusion reactions; (ao) decreased mast cell activation; (ap) decreased mast cell histamine release; (aq) decreased reduced vascular permeability; (ar) reduced edema; (as) reduced complement deposition on the graft endothelium; (at) reduced production of anaphylatoxins in the graft endothelium; (au) reduced separation of the dermal-epidermal junction; (av) reduced production of anaphylatoxins at the dermal-epidermal junction; (aw) reduced alloantibody-mediated complement activation in the graft endothelium; (ax) reduced antibody-mediated loss of the neuromuscular junction; (ay) reduced complement activation at the neuromuscular junction; (az) reduced production of anaphylatoxins at the neuromuscular junction; (ba) reduced complement deposition at the neuromuscular junction; (bb) reduced paralysis; (be) reduced numbness; (bd) increased bladder control; (be) increased bowel control; (bf) reduced mortality associated with autoantibodies; and (bg) reduced morbidity associated with autoantibodies.

[0615] In some embodiments, the antigen binding molecules of the present disclosure, when administered at one or more doses as a monotherapy or in combination therapy to an individual suffering from a complement-mediated disease or disorder, can achieve a reduction of at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% in one or more of the following outcomes, compared to the level or extent of the outcome in the individual prior to treatment with the antigen binding molecules: (a) complement activation; (b) cognitive decline; (c) neuronal loss; (d) Phospho-Tau levels in neurons; (e) glial cell activation; (f) lymphocyte infiltration; (g) macrophage infiltration; (h) antibody deposition, (i) glial cell loss; (j) oligodendrocyte loss; (k) dendritic cell infiltration; (l) neutrophil infiltration; (m) erythrocyte lysis; (n) erythrocyte phagocytosis; (o) platelet phagocytosis; (p) platelet lysis; (q) transplant rejection; (r) macrophage-mediated phagocytosis; (s) visual loss; (t) antibody-mediated complement activation; (u) autoantibody-mediated complement activation; (v) demyelination; (w) eosinophilia.

[0616] In some embodiments, the antigen binding molecules of the present disclosure, when administered at one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or condition, can achieve at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% improvement in one or more of the following outcomes: a) cognitive function; b) graft survival; c) vision; d) motor control; e) thrombosis; f) coagulation; g) renal function; and h) hematocrit (red blood cell count), compared to the level or extent of the outcome in the individual before treatment with the antigen binding molecules.

[0617] In some embodiments, administration of an antigen binding molecule of the present disclosure to an individual reduces complement activation in the individual. For example, in some embodiments, an antigen binding molecule of the present disclosure, when administered at one or more doses as a monotherapy or in combination therapy to an individual suffering from a complement-mediated disease or condition, reduces complement activation in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to complement activation in the individual before treatment with the antigen binding molecule.

[0618] In some embodiments, the use of antigen binding molecules of the present disclosure improves individual cognitive function. For example, in some embodiments, the antigen binding molecules of the present disclosure, when applied to an individual suffering from a complement-mediated disease or condition at one or more doses as a monotherapy or conjoint therapy, improve the cognitive function of the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the cognitive function of the individual before treatment with the antigen binding molecules.

[0619] In some embodiments, the antigen binding molecules of the present disclosure are used to reduce the rate of decline of individual cognitive function. For example, in some embodiments, the antigen binding molecules of the present disclosure, when applied to individuals suffering from complement-mediated diseases or conditions at one or more doses as a monotherapy or a combination therapy, reduce the rate of decline of individual cognitive function by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90% compared to the rate of decline of cognitive function of the individual before treatment with the antigen binding molecules.

[0620] In some embodiments, the antigen binding molecules of the present disclosure are administered to individuals to reduce neuronal loss in individuals. For example, in some embodiments, the antigen binding molecules of the present disclosure, when administered to individuals suffering from complement-mediated diseases or conditions at one or more doses as a monotherapy or conjoint therapy, reduce neuronal loss in individuals by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to neuronal loss in individuals before treatment with the antigen binding molecules.

[0621] In some embodiments, administering an antigen binding molecule of the present disclosure to an individual reduces the level of phospho-Tau in the individual. For example, in some embodiments, an antigen binding molecule of the present disclosure, when administered at one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or condition, reduces phospho-Tau in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the level of phospho-Tau in the individual before treatment with the antigen binding molecule.

[0622] In some embodiments, the antigen binding molecules of the present disclosure are applied to individuals to reduce the activation of glial cells in individuals. For example, in some embodiments, when the antigen binding molecules of the present disclosure are applied to individuals suffering from complement-mediated diseases or illnesses with one or more doses as monotherapy or conjoint therapy, compared with the activation of glial cells in individuals before treatment with antigen binding molecules, the activation of glial cells in individuals is reduced by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%. In some embodiments, glial cells are astrocytes or microglia.

[0623] In some embodiments, administration of an antigen binding molecule of the present disclosure to an individual reduces lymphocyte infiltration in the individual. For example, in some embodiments, an antigen binding molecule of the present disclosure, when administered to an individual with a complement-mediated disease or condition at one or more doses as a monotherapy or combination therapy, reduces lymphocyte infiltration in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%.

[0624] In some embodiments, administering an antigen binding molecule of the present disclosure to an individual reduces macrophage infiltration in the individual. For example, in some embodiments, an antigen binding molecule of the present disclosure, when administered to an individual with a complement-mediated disease or condition at one or more doses as a monotherapy or combination therapy, reduces macrophage infiltration in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%.

[0625] In some embodiments, administering an antigen binding molecule of the present disclosure to an individual reduces antibody deposition in the individual. For example, in some embodiments, an antigen binding molecule of the present disclosure, when administered at one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or condition, reduces antibody deposition in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to antibody deposition in the individual before treatment with the antigen binding molecule.

[0626] In some embodiments, administration of an antigen binding molecule of the present disclosure to an individual reduces the production of anaphylatoxins (e.g., C3a, C4a, C5a) in the individual. For example, in some embodiments, an antigen binding molecule of the present disclosure, when administered at one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or condition, reduces the production of anaphylatoxins in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, compared to the level of anaphylatoxin production in the individual prior to treatment with the antigen binding molecule.

[0627] In some embodiments, the present disclosure provides the use of an antigen binding molecule of the present disclosure or a pharmaceutical composition comprising an antigen binding molecule of the present disclosure and a pharmaceutically acceptable excipient for treating an individual suffering from a complement-mediated disease or condition. In some embodiments, the present disclosure provides the use of an anti-Cls antibody of the present disclosure for treating an individual suffering from a complement-mediated disease or condition. In some embodiments, the present disclosure provides the use of a pharmaceutical composition comprising an antigen binding molecule of the present disclosure and a pharmaceutically acceptable excipient for treating an individual suffering from a complement-mediated disease or condition.

[0628] In some embodiments, the present disclosure provides for use of an antigen binding molecule of the present disclosure in the manufacture of a medicament for treating an individual suffering from a complement-mediated disease or disorder.

[0629] In some embodiments, the present disclosure provides the use of antigen binding molecules of the present disclosure or pharmaceutical compositions comprising antigen binding molecules of the present disclosure and pharmaceutically acceptable excipients to inhibit complement activation. In some embodiments, the present disclosure provides the use of antigen binding molecules of the present disclosure or pharmaceutical compositions comprising antigen binding molecules of the present disclosure and pharmaceutically acceptable excipients to inhibit complement activation in individuals suffering from complement-mediated diseases or conditions. In some embodiments, the present disclosure provides the use of antigen binding molecules of the present disclosure to inhibit complement activation in individuals suffering from complement-mediated diseases or conditions. In some embodiments, the present disclosure provides the use of pharmaceutical compositions comprising antigen binding molecules of the present disclosure and pharmaceutically acceptable excipients to inhibit complement activation in individuals suffering from complement-mediated diseases or conditions.

[0630] In some embodiments, the disclosure provides the use of an antigen binding molecule of the disclosure in the production of a medicament for regulating complement activation. In some embodiments, the medicament inhibits complement activation. In some embodiments, the medicament inhibits complement activation in an individual suffering from a complement-mediated disease or condition.

[0631] In some embodiments, the present disclosure provides an antigen binding molecule of the present disclosure for medical treatment or a pharmaceutical composition comprising an antigen binding molecule of the present disclosure and a pharmaceutically acceptable excipient. In some embodiments, the present disclosure provides an antigen binding molecule of the present disclosure for medical treatment. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antigen binding molecule of the present disclosure and a pharmaceutically acceptable excipient for medical treatment.

[0632] In some embodiments, the present disclosure provides an antigen binding molecule of the present disclosure or a pharmaceutical composition comprising an antigen binding molecule of the present disclosure and a pharmaceutically acceptable excipient for treating an individual suffering from a complement-mediated disease or condition. In some embodiments, the present disclosure provides an antigen binding molecule of the present disclosure for treating an individual suffering from a complement-mediated disease or condition. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antigen binding molecule of the present disclosure and a pharmaceutically acceptable excipient for treating an individual suffering from a complement-mediated disease or condition.

[0633] In some embodiments, the present disclosure provides an antigen binding molecule of the present disclosure for regulating complement activation or a pharmaceutical composition comprising an antigen binding molecule of the present disclosure and a pharmaceutically acceptable excipient. In some embodiments, the present disclosure provides an antigen binding molecule of the present disclosure for regulating complement activation. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antigen binding molecule of the present disclosure and a pharmaceutically acceptable excipient for regulating complement activation. In some embodiments, the antigen binding molecule inhibits complement activation.

[0634] In another aspect, the present invention provides the use of antigen binding molecules in the production or preparation of drugs. In one embodiment, the drug is used to treat complement-mediated diseases or conditions. In a further embodiment, the drug is used in a method for treating a complement-mediated disease or condition, the method comprising administering an effective amount of the drug to an individual suffering from a complement-mediated disease or condition. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, for example, as described below. In a further embodiment, the drug is used to enhance the removal (or removal) of C1s from plasma. In a further embodiment, the drug is used to enhance the removal (or removal) of Clq, C1r and C1s complexes from plasma. In a further embodiment, the drug is used to inhibit the cleavage of complement component C4, wherein the molecule does not inhibit the cleavage of complement component C2. In a further embodiment, the drug is used to inhibit components of the classical complement pathway; in some cases, the classical complement pathway components are Cls

[0635] In a further embodiment, the medicament is for use in a method of treating an individual suffering from a complement-mediated disease or disorder, comprising administering to the individual an effective amount of the medicament.The "individual" according to any of the above embodiments may be a human.

[0636] In another aspect, the invention provides a method for treating a complement-mediated disease or condition. In one embodiment, the method comprises administering an effective amount of an antigen binding molecule to an individual suffering from such a complement-mediated disease or condition. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent to the individual, as described below. According to any of the above embodiments, an "individual" can be a person.

[0637] In another aspect, the present invention provides a method for enhancing the clearance (or removal) of C1s in individual plasma. In another aspect, the present invention provides a method for enhancing the clearance (or removal) of Clq, C1r and C1s complexes in individual plasma. In some embodiments, the antigen binding molecules of the present invention provide a method for inhibiting the cleavage of complement component C4, wherein the molecule does not inhibit the cleavage of complement component C2 in the individual. In some cases, the present invention provides a method for inhibiting the classical complement pathway components of an individual; in some cases, the classical complement pathway components are Cls. In one embodiment, the "individual" is a human.

[0638] In another aspect, the invention provides a pharmaceutical preparation comprising any antigen binding molecules provided herein, for example, for use in any of the above-mentioned methods of treatment. In one embodiment, the pharmaceutical preparation comprises any antigen binding molecules provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical preparation comprises any antigen binding molecules provided herein and at least one other therapeutic agent, for example, as described below.

[0639] The antibodies of the invention can be used alone or in combination with other agents in therapy. For example, the antibodies of the invention can be administered in combination with at least one additional therapeutic agent.

[0640] Such combination therapies described above include co-administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration (in which case administration of the molecules of the invention may be performed before, simultaneously with, and / or after administration of the additional therapeutic agent(s). In one embodiment, administration of the antigen binding molecule and administration of the additional therapeutic agent occur within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. The molecules of the invention may also be used in combination with radiotherapy.

[0641] The antigen binding molecules of the present invention (and any other therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary and intranasal, and if local treatment is required, by intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, peritoneal or subcutaneous administration. Administration can be by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various dosing regimens are contemplated herein, including but not limited to single or multiple administrations, push administration and pulse infusions at various time points.

[0642] The antigen binding molecules of the present invention will be prepared, administered and used in a manner consistent with good medical practice. Factors considered in this regard include specific conditions to be treated, specific mammals to be treated, clinical conditions of individual patients, causes of disease, delivery sites of medicaments, methods of administration, time arrangements for administration, and other factors known to doctors. The molecule is not required, but is optionally prepared together with one or more medicaments currently used to prevent or treat the condition in question. The effective amount of such other medicaments depends on the amount of the molecule present in the preparation, the type of condition or treatment, and other factors mentioned above. These are usually used with the same dosage and route of administration as described herein, or about 1 to 99% of the dosage described herein, or with experience / clinical determination of any dosage and any route suitable for use.

[0643] For the prevention or treatment of a disease, the appropriate dose of the antigen binding molecules of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and the response to the molecule, and the judgment of the attending physician. The molecule is appropriately administered to the patient at one time or in a series of treatments. Depending on the type and severity of the disease, a molecule of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg-10 mg / kg) can be an initial candidate dose for administration to the patient, for example, by one or more separate administrations, or by continuous infusion. According to the above factors, a typical daily dose can be in the range of about 1 μg / kg to 100 mg / kg or more. For repeated administration for several days or longer, depending on the patient's condition, treatment will generally continue until the desired disease symptom suppression occurs. An exemplary dose of the molecule will be in the range of about 0.05 mg / kg to about 10 mg / kg. Therefore, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered at intervals, such as once a week or once every three weeks (e.g., so that the patient receives about two to about twenty doses, or, for example, about six molecules). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0644] It will be appreciated that any of the above formulations or treatment methods may be performed using the immunoconjugates of the invention in place of or in addition to the antigen binding molecules.

[0645] H. Other methods

[0646] In one aspect, the invention provides a method for uptake of C1s or C1q into cells. In this aspect, C1s and cells are mixed in an isotonic solution. The cells express Fcγ receptors. The method comprises adding any of the above antigen binding molecules to the isotonic solution.

[0647] In one aspect, the present invention provides a method for reducing the plasma C1s and / or C1q concentration in an individual. The method comprises administering any one of the above-mentioned antigen binding molecules to the individual.

[0648] I. Products

[0649] In another aspect of the present invention, there is provided a product containing materials that can be used for treating, preventing and / or diagnosing the above-mentioned conditions. The product includes a container and a label on the container or a package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed of a variety of materials, such as glass or plastic. The container is filled with a composition, which is used in combination with another composition that effectively treats, prevents and / or diagnoses the condition, and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). At least one active ingredient in the composition is an antigen binding molecule or antibody of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. In addition, the product may include (a) a first container containing a composition, wherein the composition includes an antigen binding molecule or antibody of the present invention; and (b) a second container containing a composition, wherein the composition includes another cytotoxic agent or other therapeutic agent. The product in this embodiment of the present invention may further include a package insert, indicating that the composition can be used to treat a specific condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. From a commercial and user perspective, it may further comprise other desired materials, including other buffers, diluents, filters, needles and syringes.

[0650] It is to be understood that any of the above-described articles of manufacture may include an immunoconjugate of the invention instead of or in addition to an antigen binding molecule.

[0651] Example

[0652] IV. Examples

[0653] The following are examples of methods and compositions of the present invention.It is understood that various other embodiments may be practiced, given the general description provided above.

[0654] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patents and scientific literature cited herein are expressly incorporated by reference in their entirety.

[0655] Example 1

[0656] Preparation of recombinant C1r2s2

[0657] 1.1. Expression and purification of cynomolgus monkey C1r2s2 His / Flag tetramer

[0658] The sequences used for expression and purification were: cynomolgus monkey C1s with a C-terminal GGGGS linker and FLAG tag (SEQ ID NO: 1) and cynomolgus monkey C1r with a C-terminal GGGGS linker and 8× histidine tag. The cynomolgus monkey C1r sequence has the R463Q S654A mutation (SEQ ID NO: 2). To express the recombinant cynomolgus monkey C1r2s2 His / Flag tetramer, the FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA) was used to transiently co-express cynomolgus monkey C1s-Flag and cynomolgus monkey C1r-His. Conditioned medium expressing recombinant cynomolgus monkey C1r2s2 His / Flag tetramer was applied to anti-FlagM2 affinity resin (Sigma) and eluted with Flag peptide (Sigma). Fractions containing recombinant cynomolgus monkey C1r2s2 His / Flag tetramer were passed through an IMAC column (GE Healthcare) and eluted with an imidazole gradient. The eluted fractions containing recombinant C1r2s2 His / Flag tetramer were collected, concentrated, and subsequently placed on a Superdex 200 gel filtration column (GE Healthcare) equilibrated with 1×TBS, 2mM CaCl2 buffer. The fractions containing recombinant cynomolgus monkey C1r2s2 His / Flag were then pooled, concentrated, and stored at -80°C.

[0659] 1.2. Expression and purification of human C1r2s2 tetramer

[0660] The sequences used for expression and purification were: human C1s (NCBI reference sequence: NP_958850.1) (SEQ ID NO: 3) and human C1r (NCBI reference sequence: NP_001724.3). The human C1r sequence had a R463Q S654A mutation (SEQ ID NO: 4). To express recombinant human C1r2s2 tetramers, FreeStyle293-F cell line (Thermo Fisher, Carlsbad, CA, USA) was used to transiently co-express human C1s and human C1r. Conditioned medium expressing recombinant human C1r2s2 was applied to a HiTrap Q HP anion exchange chromatography column (GE Healthcare) and eluted with a NaCl gradient. The eluted fractions containing recombinant human C1r2s2 tetramers were collected, concentrated, and subsequently placed in a Superdex 200 gel filtration column (GE Healthcare) equilibrated with 1×TBS, 2 mM CaCl2 buffer. Fractions containing recombinant human C1r2s2 tetramer were then pooled, concentrated and stored at -80°C.

[0661] Example 2

[0662] 2.1 Preparation of cynomolgus monkey Fcγ receptor (cynomolgus monkey FcγR)

[0663] Using methods known to those skilled in the art, the synthesis of the extracellular domain gene of the cynomolgus FcγR was constructed by cloning the cDNA of each FcγR from the cynomolgus monkey. The amino acid sequence of the constructed FcγR extracellular domain is shown in the sequence list: (for cynomolgus FcγRIa, SEQ ID NO: 5; for cynomolgus FcγRIIa1, SEQ ID NO: 6; for cynomolgus FcγRIIa2, SEQ ID NO: 7; for cynomolgus FcγRIIa3, SEQ ID NO: 8; for cynomolgus FcγRIIb, SEQ ID NO: 9; for cynomolgus FcγRIIIa (R), SEQ ID NO: 10; for cynomolgus FcγRIIIa (S), SEQ ID NO: 11). Then His-tag was added to their C-termini and each gene obtained was inserted into an expression vector designed for mammalian cell expression. The target protein is expressed in the FreeStyle293 cell (Invitrogen) that expression vector is imported into human embryonic kidney cell source.After cultivation, in principle, the culture supernatant obtained by filtering and purifying by the following four steps.The first step uses SP Sepharose FF to carry out cation exchange chromatography, the second step carries out affinity chromatography (HisTrap HP) for His-tag, the third step carries out gel filtration column chromatography (Superdex200), and the fourth step is aseptic filtration.The absorbance of purified protein at 280 nm is measured by spectrophotometer, and the concentration of purified protein is determined by the extinction coefficient calculated by PACE method (ProteinScience 4:2411-2423 (1995)).

[0664] Example 3

[0665] Preparation of anti-C1s antibody

[0666] 3.1. Generation of COS0098pHv1 and preparation of COS0098pHv1-SG1077 and COS0098pHv1-SG1

[0667] The variable regions of some anti-C1s antibodies COS0098bb (VH, SEQ ID NO: 12; VL, SEQ ID NO: 13, as described in PCT / JP2018 / 042054) were humanized to reduce the potential immunogenicity of the antibodies. The complementarity determining regions (CDRs) of the anti-C1s rabbit antibody were transplanted onto the homologous human antibody framework (FR) using conventional CDR transplantation methods (Nature 321: 522-525 (1986)). Genes encoding humanized VH were synthesized and combined with human IgG1 CH (SG1, SEQ ID NO: 14), modified human IgG1 CH such as SG1077 (SEQ ID NO: 15) and SG1148 (SEQ ID NO: 16). Genes encoding humanized VL were synthesized and combined with human CL (SK1, SEQ ID NO: 26) and modified human CL (KOMC, SEQ ID NO: 17). Those combined sequences were cloned into expression vectors.

[0668] Many mutations and combinations of mutations were examined to identify mutations and combinations of mutations that improved the binding properties of the lead antibody. Multiple mutations were then introduced into the humanized variable region to enhance the binding affinity to C1s (human C1s or C1r2s2 or cynomolgus monkey C1r2s2 His / Flag) at neutral pH or to reduce the binding affinity to C1s at acidic pH. One of the optimized variants, COS0098pHv1 (VH, SEQ ID NO: 18; VL, SEQ ID NO: 19; HCDR1, SEQ ID NO: 20; HCDR2, SEQ ID NO: 21; HCDR3, SEQ ID NO: 22; LCDR1, SEQ ID NO: 23; LCDR2, SEQ ID NO: 24; and LCDR3, SEQ ID NO: 25), was thus generated from COS0098bb. COS0098pHv1-SG1077 (VH of COS0098pHv1; SEQ ID NO: 18 combined with SG1077; SEQ ID NO: 15 for the heavy chain, and VL of COS0098pHv1; SEQ ID NO: 19 combined with SK1; SEQ ID NO: 26 for the light chain) was used for in vivo PK studies in cynomolgus monkeys. COS0098pHv1-SG1 (VH of COS0098pHv1; SEQ ID NO: 18 combined with SG1; SEQ ID NO: 14 for the heavy chain, and VL of COS0098pHv1; SEQ ID NO: 19 combined with SK1; SEQ ID NO: 26 for the light chain) was used as a control antibody in cynomolgus monkey FcγR binding assays.

[0669] Antibodies were expressed in HEK293 cells co-transfected with a mixture of heavy and light chain expression vectors and purified by protein A. Gel filtration was performed further if necessary.

[0670] Example 4

[0671] Binding characterization of anti-C1s antibodies

[0672] 4.1. Evaluation of pH dependence and cross-reactivity checks

[0673] Kinetic parameters of anti-C1s antibodies against recombinant human C1s (as described in PCT / JP2018 / 042054), human C1r2s2, and cynomolgus monkey C1r2s2 His / Flag (also referred to as cynomolgus monkey C1r2s2 in the tables and figures) were evaluated at pH 7.4 and pH 5.8 at 37°C using a BIACORE (registered trademark) T200 instrument (GE Healthcare) or BIACORE 4000 (GE Healthcare). ProA / G (Pierce) was immobilized on a CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the recommended settings of GE Healthcare. Antibodies and analytes were diluted into the respective running buffers ACES pH 7.4 and pH 5.8 (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 1 mg / ml BSA, 1 mg / ml CMD, 0.05% Tween 20, 0.005 w / v% NaN3). Each antibody was captured to the sensor surface by ProA / G. Antibody capture levels were typically 100-200 resonance units (RU). Recombinant C1s were then injected at concentrations of 25 and 50 nM and then dissociated at pH 7.4 and pH 5.8. The surface was regenerated using 10 mM glycine-HCl, pH 1.5. Kinetic parameters under neutral pH conditions were determined by fitting the sensorgrams to a 1:1 binding model using BIACORE (registered trademark) T200 Evaluation Software version 2.0 (GE Healthcare) or BIACORE 4000 Evaluation Software. The dissociation rate at pH 5.8 was determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software. The sensorgrams for all antibodies at an antigen concentration of 50 nM are shown in Figure 2. Figure 1 and Figure 2 The association rate (ka), dissociation rate (kd), binding affinity (KD) of the antibodies at pH 7.4 and the dissociation rate (kd) at pH 5.8 are listed in Table 2.

[0674] Although COS0098bb (VH, SEQ ID NO: 12 and VL, SEQ ID NO: 13) did not clearly exhibit a faster dissociation rate at pH 5.8 than at pH 7.4, COS0098pHv1 (VH, SEQ ID NO: 18 and VL, SEQ ID NO: 19) showed a faster dissociation rate at pH 5.8 than at pH 7.4.

[0675] In addition, BIACORE (registered trademark) kinetic analysis was performed to observe the cross-reactivity of anti-C1s antibodies to human C1r2s2 and cynomolgus monkey C1r2s2 (cynomolgus monkey C1r2s2 His / Flag). The binding kinetics and affinity at pH 7.4 are listed in Table 2. All anti-C1s antibodies showed comparable KD for human C1r2s2 and cynomolgus monkey C1r2s2.

[0676] [Table 2]

[0677]

[0678] 4.2. Evaluation of Antibody Binding Activity to Cynomolgus FcγR Using Biacore

[0679] Analysis of the interaction between the antibody and the FcγR prepared as above was performed using BIACORE (registered trademark) T200 (GE Healthcare). A 50 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride and 0.05 w / v%-20 was used as the running buffer, and the measurement temperature was set to 25°C. A chip produced by fixing Protein L (BioVision) to the S series sensor chip CM4 (GE Healthcare) by an amine coupling method was used. After capturing 500 or 1000 resonance units (RU) of the target antibody on the chip, the FcγR diluted with the running buffer was allowed to interact, and their binding amount to the antibody was measured. The cynomolgus monkey FcγRIa was diluted to 8 nM, and the others were diluted to 1000 nM. Then, the antibodies captured on the chip were washed with 10 mM glycine-HCl, pH 1.5, and the chip was regenerated and reused. The binding amount of FcγR was divided by the binding amount of each captured antibody using biacore evaluation software to obtain the binding activity, and these values ​​were compared. Furthermore, the relative value of the binding activity of COS0098pHv1-SG1077 to each FcγR relative to COS0098pHv1-SG1 (an antibody having the sequence of naturally occurring human IgG1 at positions 234 to 239, 265 to 271, 295, 296, 298, 300, and 324 to 337 according to EU numbering) was calculated.

[0680] Table 3 shows the binding activity values ​​of the antibodies and the relative amounts of COS0098pHv1-SG1077 and COS0098pHv1-SG1. The binding activity values ​​of COS0098pHv1-SG1077 to various cynomolgus monkey FcγRIIa and FcγRIIb were higher than those of COS0098pHv1-SG1. Compared with COS0098pHv1-SG1, the binding activity of COS0098pHv1-SG1077 to FcγRIIb was increased by 3.26 times, and the binding activity to various cynomolgus monkey FcγRIIa was increased by 2.57-5.74 times.

[0681] [Table 3]

[0682]

[0683] Example 5

[0684] 5.1 PK study in cynomolgus monkeys

[0685] Measurement of total cynomolgus C1s in monkey plasma by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC / ESI-MS / MS) and C1q concentration

[0686] method

[0687] Total cynomolgus C1s and C1q concentrations in cynomolgus monkey plasma were measured by high performance liquid chromatography-electrospray tandem mass spectrometry (LC / ESI-MS / MS).

[0688] Sample preparation

[0689] The total concentration of cynomolgus monkey C1s in plasma was measured by LC / ESI-MS / MS. Calibration standards were prepared by mixing and diluting cynomolgus monkey C1s in mouse plasma at specified amounts, yielding cynomolgus monkey C1s concentrations of 0.954, 1.91, 3.82, 7.63, 15.3, 30.5, and 61.1 μg / mL. Cynomolgus monkey plasma samples were diluted 5-fold with mouse plasma. 2 μL of calibration standards or diluted plasma samples were mixed with 26.5 μL of mixed reagent in 50 mmol / L ammonium bicarbonate (7.5 mol / L urea / 100 mmol / L dithiothreitol / 10 μg / mL lysozyme (hen egg white) / 300 μg / mL human C1s = 20 / 2 / 2.5 / 2) and incubated at 56°C for 45 min. Then, 2 μL of 500 mmol / L iodoacetamide was added and incubated at 37°C in the dark for 30 min. Next, 160 μL of 0.5 μg / mL sequencing grade modified trypsin (Promega) in 50 mmol / L ammonium bicarbonate was added and incubated overnight at 37°C. Finally, 5 μL of 10% trifluoroacetic acid was added to inactivate any residual trypsin. 50 μL of the digested sample was analyzed by LC / ESI-MS / MS.

[0690] The concentration of cynomolgus monkey C1q in plasma was measured by LC / ESI-MS / MS. Calibration standards were prepared by mixing and diluting cynomolgus monkey C1q in human plasma at defined amounts, yielding cynomolgus monkey C1q concentrations of 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, and 100 μg / mL. Cynomolgus monkey plasma samples were diluted 5-fold with human plasma. 2 μL of calibration standards and diluted plasma samples were mixed with 40 μL of anti-human C1q (Cell Sciences), and anti-C1s antibody in 1% bovine serum albumin was applied separately at 25°C for 1.5 hours. Each sample was mixed with 50 μL of 10-fold diluted protein A magnetic beads (Protenova) in PBS containing 0.05% Tween-20 at 25°C for 1.5 hours. After washing three times with PBS containing 0.05% Tween-20, the samples were mixed with 26.5 µL of a mixed reagent in 50 mmol / L ammonium bicarbonate (7.5 mol / L urea / 100 mmol / L dithiothreitol / 10 µg / mL lysozyme (hen egg white) = 20 / 2 / 2.5) and incubated at 56°C for 45 min. Then, 2 µL of 500 mmol / L iodoacetamide was added and incubated at 37°C in the dark for 30 min. Next, 160 µL of 0.5 μg / mL sequencing grade modified trypsin (Promega) in 50 mmol / L ammonium bicarbonate was added and incubated overnight at 37°C. Finally, 5 µL of 10% trifluoroacetic acid was added to inactivate any residual trypsin. 50 µL of the digested sample was analyzed by LC / ESI-MS / MS.

[0691] LC / ESI-MS / MS analysis

[0692] LC / ESI-MS / MS was performed using a Xevo TQ-S triple quadrupole instrument (Waters) equipped with a 2D I-class UPLC (Waters). The cynomolgus monkey C1s, human C1s, and cynomolgus monkey C1q specific peptides LLEVPEAR, LLEVPEGR, and YQSVFTVAR were monitored by selected reaction monitoring (SRM), respectively. The SRM transitions were [M+2H] 2+ (m / z 463.8 to y6 ion (m / z 700.3)), [M+2H] 2+ (m / z 456.8 to y6 ion (m / z 686.4)), and [M+2H] 2+ (m / z 535.8 to y7 ion (m / z 779.4)). Each calibration curve was constructed by weighted (1 / x2) linear regression using peak areas plotted against concentration. The concentration in cynomolgus monkey plasma was calculated from the calibration curve using the analytical software Masslynx Ver. 4.1 (Waters).

[0693] Pharmacokinetic evaluation of total C1s and C1q after administration of anti-C1s antibody in cynomolgus monkeys

[0694] The in vivo pharmacokinetics of C1s and C1q in cynomolgus monkeys were evaluated following administration of COS0098pHv1-SG1077 to cynomolgus monkeys.Three male monkeys were assigned to each dosing group.

[0695] COS0098pHv1-SG1077 was injected intravenously into monkeys at a dose of 10 mg / kg on day 0. The dose setting of COS0098pHv1-SG1077 was adjusted to an excess concentration of physiological plasma cynomolgus monkey C1s and C1q concentrations after injection. Blood was collected at -1 day before injection and 5 minutes, 2, 8 hours, 1, 2, 4, 7, 14, 21 and 28 days after injection.

[0696] These blood samples were immediately centrifuged to separate plasma samples. Plasma concentrations of cynomolgus monkey C1s and C1q were measured by LC / ESI-MS / MS at each sampling point.

[0697] SG1077 contains mutations that enhance Fcγ receptor binding. After injection of COS0098pHv1-SG1077, plasma concentrations of C1s in cynomolgus monkeys decreased to less than 30% of baseline ( Figure 3 Similarly, after injection of COS0098pHv1-SG1077, plasma concentrations of C1q in cynomolgus monkeys decreased to less than 20% of baseline ( Figure 4 All plasma C1q concentration samples on days 4, 7, and 14 after the first injection were below the limit of quantification (days 4 and 7: 9.75 μg / mL, day 14: 19.5 μg / mL). This result suggests that COS0098pHv1-SG1077 has a strong potential to accelerate the elimination of C1s and C1q.

Claims

1. An antigen-binding molecule that binds to C1s and comprises a heavy chain variable region, a light chain variable region, and an Fc region, wherein (i) the heavy chain variable region and / or the light chain variable region comprises at least one amino acid, wherein the at least one amino acid is capable of increasing the ratio of the KD value of the antigen-binding molecule with C1s in the acidic pH range to the KD value of the antigen-binding molecule with C1s in the neutral pH range, KD (acidic pH range) / KD (neutral pH range), compared to a first reference antibody comprising two heavy chain variable regions and two light chain variable regions, wherein the heavy chain variable region in the first reference antibody comprises the amino acid sequence of SEQ ID NO: 12 and the light chain variable region in the first reference antibody comprises the amino acid sequence of SEQ ID NO: 13; (ii) the Fc region comprises at least one amino acid, wherein the at least one amino acid is capable of increasing the ability of the antigen-binding molecule to bind to FcRn in an acidic pH range compared to a second reference antibody comprising a paired Fc region, wherein the Fc region in the second reference antibody comprises the amino acid sequence of SEQ ID NO: 14; and (iii) the Fc region comprises at least one amino acid, wherein the at least one amino acid can increase the binding ability of the antigen-binding molecule to the Fcγ receptor in the neutral pH range compared to the second reference antibody.

2. The antigen-binding molecule of claim 1, wherein the heavy chain variable region and the light chain variable region comprise at least one amino acid that can increase the stability of the antigen-binding molecule compared to the first reference antibody.

3. The antigen binding molecule of claim 1, wherein the antigen binding molecule comprises a constant region, wherein the Fc region of the antigen binding molecule is in the constant region and the constant region comprises at least one amino acid that can reduce the immunogenicity of the antigen binding molecule. The antigen-binding molecule of claim 1 , wherein the Fc region of the antigen-binding molecule comprises at least one amino acid capable of reducing binding activity to rheumatoid factor.

5. The antigen-binding molecule of claim 1, wherein the variable region in (i) comprises at least one of the following amino acids: The histidine at position 27 in the heavy chain variable region, Proline at position 59 of the heavy chain variable region and Histidine at position 96 of the light chain variable region (All numbering is according to the Kabat numbering system).

6. The antigen-binding molecule of claim 1, wherein the Fc region in (ii) comprises at least one amino acid selected from the group consisting of leucine at position 428, alanine at position 434, and threonine at position 436 (all numbering is according to the EU numbering system).

7. The antigen-binding molecule of claim 1, wherein the Fc region in (iii) comprises at least one of the following amino acids: Tyrosine at position 234, Tryptophan at position 235, Asparagine at position 236, Aspartic acid at position 238, Valine at position 250, Isoleucine at position 264, Aspartic acid at position 268, Leucine at position 295, Proline at position 307, Threonine at position 326 and Lysine at position 330 (All numbers are according to the EU numbering system).

8. The antigen-binding molecule of claim 1, wherein the Fc region in (iii) comprises the following amino acids (a) or (b); (a) tryptophan at position 235, asparagine at position 236, aspartic acid at position 268, leucine at position 295, threonine at position 326, and lysine at position 330, or (b) tyrosine at position 234, aspartic acid at position 238, valine at position 250, isoleucine at position 264, proline at position 307, and lysine at position 330 (All numbers are according to the EU numbering system).

9. The antigen binding molecule of claim 2, wherein the at least one amino acid capable of increasing stability is one or both of alanine at position 96 of the heavy chain variable region and glutamic acid at position 53 of the light chain variable region (both numberings are according to the Kabat numbering system).

10. The antigen binding molecule of claim 3, wherein the at least one amino acid capable of reducing immunogenicity is arginine at position 214 according to the EU numbering system.

11. The antigen-binding molecule of claim 4, wherein the at least one amino acid capable of reducing binding activity to rheumatoid factor is one or both of arginine at position 438 and glutamic acid at position 440 (both numberings are according to the EU numbering system).

12. An antigen-binding molecule that competes with an antibody for binding to C1s, wherein the antibody comprises: a heavy chain variable region comprising HVR-H1 of SEQ ID NO: 20, HVR-H2 of SEQ ID NO: 21, and HVR-H3 of SEQ ID NO: 22, and a light chain variable region comprising HVR-L1 of SEQ ID NO: 23, HVR-L2 of SEQ ID NO: 24, and HVR-L3 of SEQ ID NO:

25. The antigen binding molecule of claim 1 , wherein the heavy chain variable region and the light chain variable region comprise human frameworks.

14. An antigen-binding molecule comprising one or both of a VH region comprising 95% identity with the amino acid sequence of SEQ ID NO: 18 and a VL region comprising 95% identity with the amino acid sequence of SEQ ID NO:

19.

15. An antigen-binding molecule comprising: a heavy chain variable region comprising HVR-H1 of SEQ ID NO: 20, HVR-H2 of SEQ ID NO: 21, and HVR-H3 of SEQ ID NO: 22, and one or both of a light chain variable region comprising HVR-L1 of SEQ ID NO: 23, HVR-L2 of SEQ ID NO: 24, and HVR-L3 of SEQ ID NO:

25.

16. An antigen-binding molecule that binds to C1s and does not compete with C1s for binding to C1q. The antigen-binding molecule according to claim 16 , which is the antigen-binding molecule according to any one of claims 1 to 15 .

18. An antigen-binding molecule, the epitope of which is located in the CCP-SP domain of C1s. The antigen-binding molecule according to claim 18 , which is the antigen-binding molecule according to any one of claims 1 to 16 .

20. An antigen binding molecule that binds to C1s and is capable of reducing the plasma C1q concentration in a subject to 20% or less of the subject's baseline. The antigen-binding molecule according to claim 20 , which is the antigen-binding molecule according to any one of claims 1 to 16 and 18.

22. The antigen-binding molecule of claim 20, wherein the individual is a cynomolgus monkey.

23. The antigen-binding molecule of claim 20, wherein the dose of the antigen-binding molecule is 10 mg / kg.

24. A pharmaceutical composition comprising the antigen-binding molecule of any one of claims 1 to 16, 18 and 20 and a pharmaceutically acceptable carrier.

25. The pharmaceutical composition of claim 24 for use in treating a subject suffering from a complement-mediated disease or disorder.

26. A method for taking up C1s or C1q into cells, wherein the C1s and the cells are mixed in an isotonic solution, the cells express Fcγ receptors, and the method comprises adding the antigen-binding molecule of any one of claims 1 to 16, 18 and 20 to the isotonic solution.

27. Use of an antigen binding molecule according to any one of claims 1 to 16, 18 and 20 for the preparation of a medicament for reducing plasma C1s and / or C1q concentration in an individual.

28. Use of an antigen binding molecule as claimed in any one of claims 1 to 16, 18 and 20 in the preparation of a medicament for treating a subject suffering from a complement-mediated disease or disorder.

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