Antigen binding molecule, pharmaceutical composition and method

Abstract

To provide an antigen binding molecule which binds to a C1s, and includes a heavy chain variable region, a light chain variable region and Fe region.SOLUTION: An antigen binding molecule binds to a C1s, and includes: a heavy chain variable region; a light chain variable region; and a Fc region. The antigen binding molecule is configured so that: (i) the heavy chain variable region and / or the light chain variable region include at least one amino acid, the amino acid capable of increasing a KD (acidic pH range) / KD (neutral pH range) which is a ratio of a KD value of the antigen binding molecule to the C1s in the acidic pH range with respect to a KD value of the antigen binding molecule to C1s in a neutral pH region, relative to that of the first reference antibody; (ii) the Fc region includes at least one amino acid which can increase a binding capacity of the antigen binding molecule to FcRn in the acidic pH range relative to that in a second reference antibody; (iii) the Fc region includes at least one amino acid which can increase a binding capacity of the antigen binding molecule to a Fcγ receptor in the neutral pH range, relative to that in the second reference antibody.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to antigen-binding molecules, pharmaceutical compositions, and methods. [Background technology]

[0002] The complement system contains about 25-30 complement proteins that play a key role in the host's defense against pathogens, foreign antigens, and tumor cells. The complement system is also involved in maintaining homeostasis by removing immune complexes and apoptotic cells from the body. Complement components carry out their functions by interacting in a cascade of enzymatic processes and membrane-binding events. The end result of these processes is the generation of products with lytic, immunomodulatory, and opsonic functions.

[0003] It is widely known that the complement system can be categorized into three distinct pathways: the classical pathway, the reticular pathway, and the alternative pathway. Although the initiation of each pathway is different, all three pathways share the same terminal complement components (C5-C9) that converge and are ultimately responsible for the destruction of target cells.

[0004] The C1 complex is a large protein complex that functions as a major initiator of the classical pathway cascade. The C1 complex is composed of three components, C1q, C1r, and C1s, in a molar ratio of 1:2:2, respectively (Non-Patent Document 1). The classical pathway is initiated when the C1 complex binds to an antibody-bound target. C1q, which has six globular heads, mediates the binding of the C1 complex to the antibody through an avidity-type interaction with the Fc region. Upon tight binding to the target, C1r in the C1 complex autoactivates and becomes enzymatically active. The activated C1r then cleaves and activates the proenzyme C1s in the C1 complex (Non-Patent Document 2). The activated C1s then cleaves its substrates, the complement components C2 and C4, into C2a / C2b and C4a / C4b fragments, respectively. This allows the assembly of C4b2a, a C3 convertase, to appear on the surface of the target, which cleaves C3 to form C3b. C3b then cleaves C5, initiating the formation of the terminal membrane attack complex of C5b, C6, C7, C8, and C9, which lyses the target through pore formation.

[0005] C1s forms homodimers in a calcium-dependent manner (Non-Patent Document 3). It has been reported that at a calcium ion concentration of 1 mM, most of C1s is in a dimeric state, and at a calcium concentration of 1 nM, it is mainly in a monomeric state (Non-Patent Document 4). In blood, C1s and C1r are mostly bound together as calcium-dependent C1r2s2 heterotetramers, which reversibly bind to C1q in a 1:1 ratio to form the C1 complex. In the absence or low concentration of calcium, the C1r2s2 tetramer dissociates into one C1r dimer and two C1s monomers (Non-Patent Document 5). C1s is a 79 kDa glycoprotein, and 5-6% of its mass is due to glycosylation (Non-Patent Document 6). The concentration of C1s in serum has been reported to be approximately 55 μg / mL (0.7 μM) (Non-Patent Document 7).

[0006] Although a properly functioning complement system protects the host from pathogens, dysregulation or inappropriate activation of the classical pathway leads to a variety of complement-mediated disorders, including, but not limited to, autoimmune hemolytic anemia (AIHA), Behcet's disease, bullous pemphigoid (BP), immune thrombocytopenic purpura (ITP), etc. Thus, inhibition of excessive or uncontrolled activation of the classical pathway may provide clinical benefit to patients with such disorders.

[0007] Antibodies are very attractive pharmaceutical agents because they are stable in plasma, highly specific for their targets, and generally exhibit good pharmacokinetic profiles. However, due to their large molecular size, the doses of therapeutic antibodies are usually high. If the target is present in large amounts, the therapeutic dose of the antibody required will be even higher. As a result, methods to improve the pharmacokinetics, pharmacodynamics, and antigen-binding properties of antibodies are attractive ways to reduce the dose and high manufacturing costs associated with therapeutic antibodies.

[0008] Several previous reports have described anti-C1s antibodies. For example, Matsumoto et al. (1986) (Non-Patent Document 8) described three antibodies that bind to different epitopes on C1s. One clone bound preferentially to the active form of C1s, while the other two bound to both the proenzyme and active forms of C1s. Only one of these two clones was able to inhibit the cleavage of C2 and C4 by C1s. Patent Document 1 describes an antibody that inhibits the cleavage of C4 through C1s but not that of C2. In addition, Patent Document 2 describes several antibodies that bind to conformational epitopes on C1s that are selective for the active form of C1s compared to the proenzyme form. Patent Document 3 describes two clones of anti-C1s antibodies that can block the cleavage of C4.

[0009] The affinity of an antibody to its antigen determines how effectively it can neutralize its target. To increase the affinity of an antibody so as to reduce the dose required for therapeutic effect, various affinity maturation methods (Non-Patent Document 9) have been used. However, one antibody molecule typically has two binding sites, and therefore is limited in that it can only neutralize two targets (one antigen per binding site) after administration. Even if an antibody can bind to a target with infinite affinity through covalent interactions, the maximum number of targets neutralized by the antibody is still limited to two.

[0010] 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 neutralization of multiple antigen molecules by a single antibody molecule (Non-Patent Document 10, Patent Document 4). pH-dependent antibodies bind strongly to their antigens under neutral pH conditions in plasma, but dissociate from the antigens under acidic pH conditions in the endosomes of cells. Upon dissociation from the antigen, the antibody is recycled into plasma by the FcRn receptor, and the dissociated antigen is degraded in the lysosomes of cells. The recycled antibody is then free to bind to and neutralize antigen molecules again, and this process is repeated as long as the antibody remains in the blood. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2014 / 071206 [Patent Document 2] WO2014 / 066744 [Patent Document 3] WO2014 / 186599 [Patent Document 4] WO2009 / 125825 [Non-patent literature]

[0012] [Non-Patent Document 1] Wang et. al. Mol Cell. 2016 Jul 7;63(1):135-45 [Non-Patent Document 2] Mortensen et. al. Proc Natl Acad Sci US A. 2017 Jan 31;114(5):986-991 [Non-Patent Document 3] Arlaud et. al. Biochim Biophys Acta. 1980 Nov 6;616(1):105-15 [Non-Patent Document 4] Rivas et. al. Biochemistry. 1992 Dec 1;31(47):11707-12 [Non-Patent Document 5] Rossi et.al. Methods Mol Biol. 2014;1100:43-60 [Non-Patent Document 6] Petillot et. al. FEBS Lett. 1995 Jan 30;358(3):323-8 [Non-Patent Document 7] Shi et. al. Blood. 2014 Jun 26;123(26):4015-22 [Non-Patent Document 8] Matsumoto et. al. J Immunol. 1986 Nov 1;137(9):2907-12 [Non-Patent Document 9] Kim et. al. Methods Mol Biol. 2014;1131:407-20 [Non-Patent Document 10] Igawa et. al. Nat Biotechnol. 2010 Nov;28(11):1203-7 Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention provides antigen-binding molecules, pharmaceutical compositions, and methods. [Means for solving the problem]

[0014] The present invention provides an antigen-binding molecule that binds to C1s and includes 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 include at least specific amino acids that can change the antigen-binding ability in a specific manner. The Fc region includes at least specific amino acids that can change its function in a specific manner.

[0015] Specifically, the present invention relates to: [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, (i) the heavy chain variable region and / or the light chain variable region comprises at least one amino acid that can increase the KD(acidic pH range) / KD(neutral pH range), which is the ratio of the KD value of the antigen-binding molecule for C1s in the acidic pH range to the KD value of the antigen-binding molecule for C1s in the neutral pH range, compared to that of a first reference antibody comprising two heavy chain variable regions and two light chain variable regions, wherein the heavy chain variable region of the first reference antibody comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region of the first reference antibody comprises the amino acid sequence of SEQ ID NO: 13; (ii) the Fc region comprises at least one amino acid that can enhance the binding ability of the antigen-binding molecule to FcRn in the acidic pH range compared to that of a second reference antibody comprising a paired Fc region, the Fc region of the second reference antibody comprising the amino acid sequence of SEQ ID NO: 14; and (iii) the Fc region comprises at least one amino acid that can enhance the binding ability of the antigen-binding molecule to an Fcγ receptor in the neutral pH range compared to that of a second reference antibody; Antigen-binding molecules. [2] The antigen-binding molecule of [1], wherein the heavy chain variable region and the light chain variable region contain at least one amino acid that can improve the stability of the antigen-binding molecule compared to that of a first reference antibody. [3] The antigen-binding molecule of [1] or [2], wherein the antigen-binding molecule comprises a constant region, an Fc region of the antigen-binding molecule is located within the constant region, and the constant region comprises at least one amino acid capable of reducing the immunogenicity of the antigen-binding molecule. [4] The antigen-binding molecule of any one of [1] to [3], wherein the Fc region of the antigen-binding molecule contains at least one amino acid capable of reducing the binding activity to a rheumatoid factor. [5] The variable region of (i) comprises the following amino acid: a histidine at position 27 in the heavy chain variable region; a proline at position 59 in the heavy chain variable region, and Histidine at position 96 in the light chain variable region (All numbers follow the Kabat numbering system) The antigen-binding molecule according to any one of [1] to [4], comprising at least one of the following: [6] The antigen-binding molecule of any one of [1] to [5], wherein the Fc region of (ii) contains 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 according to the EU numbering system). [7] The Fc region of (iii) comprises the following amino acid: Tyrosine at position 234, Tryptophan at 235th position, Asparagine at position 236, Aspartic acid at position 238; Balin, ranked 250th; Isoleucine at position 264, Aspartic acid at position 268; Leucine at 295th place; Proline at position 307; Threonine at position 326, and Lysine at position 330 (All numbers follow the EU numbering system) The antigen-binding molecule according to any one of [1] to [6], comprising at least one of the following: [8] The Fc region of (iii) comprises the following amino acid (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 follow the EU numbering system) The antigen-binding molecule according to any one of [1] to [7], [9] The antigen-binding molecule of [2], wherein the at least one amino acid capable of improving stability is either or both of alanine at position 96 in the heavy chain variable region and glutamic acid at position 53 in the light chain variable region (both numbers according to the Kabat numbering system).

[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.

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

[12] An antigen-binding molecule that competes with an 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, for binding to C1s.

[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 human-derived frameworks.

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

[15] An antigen-binding molecule comprising either or both of a heavy chain variable region comprising HVR-H1 of SEQ ID NO: 20, HVR-H2 of SEQ ID NO: 21, or 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, or HVR-L3 of SEQ ID NO: 25.

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

[17] An antigen-binding molecule according to

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

[15] .

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

[19] An antigen-binding molecule according to

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

[17] .

[20] An antigen-binding molecule that binds to C1s and can reduce plasma C1q concentration in an individual to 20% or less of the individual's baseline.

[21] An antigen-binding molecule according to

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

[19] .

[22] The antigen-binding molecule of

[20] or

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

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

[20] to

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

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

[23] and a pharma- ceutically acceptable carrier.

[25] The pharmaceutical composition of

[24] for use in treating an individual having a complement-mediated disease or disorder.

[26] A method for incorporating C1s or C1q into cells, comprising mixing C1s and the cells in an isotonic solution, the cells expressing Fcγ receptors, and adding any one of the antigen-binding molecules of [1] to

[23] to the isotonic solution.

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

[23] .

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

[23] . [Brief description of the drawings]

[0016] [Figure 1] 1 shows BIACORE® sensorgrams showing the interaction of COS0098bb with human C1r2s2 and cynomolgus C1r2s2 to evaluate pH dependency and cross-reactivity to cynomolgus C1r2s2 and human C1r2s2, as described in Example 4.1. The sensorgrams were obtained by injecting human C1r2s2 (solid line, dashed line) and cynomolgus C1r2s2 (dotted line, long dashed line) onto the sensor surface immobilized with COS0098bb. The antibody / antigen complex formed at pH 7.4 was dissociated at pH 7.4 and then further dissociated at pH 5.8 (the conditions are shown as pH 7.4-7.4-5.8), and the antibody / antigen complex formed at pH 5.8 was dissociated at pH 5.8 (the conditions are shown as pH 5.8-5.8) (Example 4). [Diagram 2] 2 shows BIACORE® sensorgrams showing the interaction of COS0098pHv1 with human C1r2s2 and cynomolgus C1r2s2 to evaluate pH dependency and cross-reactivity to cynomolgus C1r2s2 and human C1r2s2, as described in Example 4.1. The sensorgrams were obtained by injecting human C1r2s2 (solid line, dashed line) and cynomolgus C1r2s2 (dotted line, long dashed line) onto the sensor surface immobilized with COS0098pHv1. The antibody / antigen complex formed at pH 7.4 was dissociated at pH 7.4 and then further dissociated at pH 5.8 (the conditions are shown as pH 7.4-7.4-5.8), and the antibody / antigen complex formed at pH 5.8 was dissociated at pH 5.8 (the conditions are shown as pH 5.8-5.8) (Example 4). [Diagram 3]FIG. 3 is a graph showing the removal of plasma C1s by COS0098pHv1-SG1077R or IPN009VH2VK3-SG4GK (Example 5). [Figure 4] FIG. 4 is a graph showing the removal of plasma C1q by COS0098pHv1-SG1077R or IPN009VH2VK3-SG4GK (Example 5). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The techniques and procedures described or cited herein are generally well understood and may be found in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY;Current Protocols in Molecular Biology (FM Ausubel, et al. eds., (2003));the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A 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 Notebook (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; 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 C.C.Blackwell, eds.);Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987);PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994);Current Protocols in Immunology (JE Coligan et al., eds., 1991);Short Protocols in Molecular Biology (Wiley and Sons, 1999);Immunobiology (CA Janeway and P. Travers, 1997);Antibodies (P. Finch, 1997);Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989);Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The 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 Company, 1993), using conventional techniques commonly employed by those of skill in the art.

[0018] I. Definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, NY 1992) provide those skilled in the art with general guidance for many of the terms used in this application. All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

[0019] For purposes of interpreting this specification, the following definitions shall apply and whenever applicable, terms used in the singular shall include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. In the event that any of the definitions below conflict with any document incorporated herein by reference, the definition below shall control.

[0020] An "acceptor human framework" for the purposes of this specification is a framework that comprises the 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 may comprise those same amino acid sequences or may 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 the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0021] "Affinity" refers to the strength of the total non-covalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used herein refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by a dissociation constant (Kd or KD). Affinity can be measured by conventional methods known in the art, including those described herein. Individual specific illustrative and exemplary embodiments for measuring binding affinity are described below. The term "binding activity" refers to the strength of the total 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). As used herein, binding activity is not strictly limited to activity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). When the members of a binding pair can bind to each other in both monovalent and multivalent binding manners, avidity is the strength of the sum of these bonds. The avidity of molecule X to its partner Y can usually be expressed by dissociation constant (KD). Alternatively, the binding rate and dissociation rate (Kon and Koff) can be used to evaluate binding. Avidity 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.

[0022] An "affinity matured" antibody refers to an antibody with one or more modifications in one or more hypervariable regions (HVRs) that result in improved affinity of the antibody for antigen, compared to a parent antibody that does not possess the modifications.

[0023] The term "anti-C1s antibody" or "antibody that binds to C1s" refers to an antibody that can bind to C1s with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent when targeted to 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 that binds to C1s has a binding affinity 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 less, e.g. 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 M) In certain embodiments, the anti-C1s antibody binds to an epitope of C1s that is conserved among C1s from different species.

[0024] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0025] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete 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.

[0026] 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 own antigen by 50% or more in a competitive assay, and conversely, the reference antibody blocks the binding of the antibody to its own antigen by 50% or more in a competitive assay. Exemplary competitive assays are provided herein.

[0027] 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.

[0028] The "class" of an antibody refers to the type of constant domain or constant region present in the antibody's heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0029] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes the death or destruction of cells. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212Pb, and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, 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, for example, 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, as disclosed below.

[0030] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include: C1q 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 receptor); and B cell activation.

[0031] An "effective amount" of an agent (eg, a pharmaceutical formulation) refers to the amount, at dosages and for periods of time necessary, effective to achieve the desired therapeutic or prophylactic result.

[0032] The term "epitope" includes any determinant that can be bound by an antibody. An epitope is a region of an antigen that is bound by an antibody that targets the antigen and includes specific amino acids that directly contact the antibody. Epitopes can include chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. In general, an antibody specific for a particular target antigen preferentially recognizes an epitope on that target antigen in a complex mixture of proteins and / or macromolecules.

[0033] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that includes at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain, with the exception that the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. 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 Ed. Public Health Service, National Institutes of Health, Bethesda, MD 1991.

[0034] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain typically consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences typically appear in VH (or VL) in the following order: FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0035] The terms "full length antibody," "complete antibody," and "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0036] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the originally transformed cell and progeny derived from that cell regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Also included herein are mutant progeny that have the same function or biological activity as that for which the original transformed cell was screened or selected.

[0037] A "human antibody" is an antibody with an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell, or derived from a human antibody repertoire or other non-human source that uses human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0038] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Usually, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Usually, the subgroup of sequences is a subgroup in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3. In one embodiment, for VL, the subgroup is subgroup kappa I according to Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III according to Kabat et al., supra.

[0039] A "humanized" antibody refers to a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody contains substantially all of at least one, and typically two, variable domains, in which all or substantially all HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all FRs correspond to those of a human antibody. A humanized antibody may optionally contain 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 an antibody that has undergone humanization.

[0040] The term "hypervariable region" or "HVR" as used herein refers to each region of an antibody variable domain that is hypervariable in sequence (the "complementarity determining region" or "CDR") and / or forms structurally defined loops (the "hypervariable loops") and / or contains antigen contact residues (the "antigen contacts"). Typically, antibodies contain six HVRs: three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Exemplary HVRs herein include the following: (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)); (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., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (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 (d) combinations 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). Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0041] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to, cytotoxic agents.

[0042] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0043] An "isolated" antibody is one that has been separated from the components of its original environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as measured by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0044] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its original environment. Isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0045] "Isolated nucleic acid encoding an anti-C1s antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including nucleic acid molecules carried on a single vector or separate vectors, and including nucleic acid molecules present in one or more locations in a host cell.

[0046] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population are identical and / or bind to the same epitope, except for possible variant antibodies (e.g., variant antibodies containing naturally occurring mutations or that arise during the manufacture of a monoclonal antibody preparation, such variants are usually present in small amounts). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals that contain all or part of the human immunoglobulin loci; such methods and other exemplary methods for making monoclonal antibodies are described herein.

[0047] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in a pharmaceutical formulation.

[0048] "Native antibodies" refer to immunoglobulin molecules with various structures that occur in nature. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or the 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 called the variable light domain or the light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain.

[0049] The term "package insert" is used to refer to instructions typically included in commercial packaging of a therapeutic product that contain information about the indications, usage, dosage, method of administration, concomitant therapy, contraindications, and / or warnings regarding the use of such therapeutic product.

[0050] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences to obtain the maximum percent sequence identity and introducing gaps, if necessary, and not considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved by various methods within the skill 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 appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0051] The ALIGN-2 sequence comparison computer program is the copyright of Genentech, Inc., and its source code has been filed with user documentation in the US Copyright Office, Washington DC, 20559, where it is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program is compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, it can be said that a given amino acid sequence A has or contains a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in its alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0052] The term "pharmaceutical formulation" refers to a preparation in a form such that the biological activity of the active ingredients contained therein can be exerted, and which does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered.

[0053] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0054] 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), unless otherwise indicated. The term encompasses "full-length" unprocessed C1s as well as any form of C1s resulting from processing in a cell. The term also encompasses naturally occurring variants of C1s, such as splice variants and allelic variants. An exemplary amino acid sequence of human C1s is shown in SEQ ID NO:3. An exemplary amino acid sequence of cynomolgus monkey C1s is shown in SEQ ID NO:1.

[0055] As used herein, "treatment" (and its grammatical derivatives, such as "treat", "treating", etc.) refers to a clinical intervention intended to modify the natural course of the individual being treated, and may be performed prophylactically or during the course of a clinical condition. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, attenuation of any direct or indirect pathological effects of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay disease onset or slow disease progression.

[0056] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies usually have a similar structure, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). (See, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007).) One VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated by screening a complementary library of VL or VH domains, respectively, with a VH or VL domain from an antibody that binds to the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0057] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the genome of a host cell into which it is introduced. A vector can effect expression of a nucleic acid to which it is operatively linked. Such vectors are also referred to herein as "expression vectors."

[0058] II. Antigen-binding molecules 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.

[0059] In one embodiment, the heavy chain variable region and / or the light chain variable region comprises at least one amino acid that can increase the KD(acidic pH range) / KD(neutral pH range), which is the ratio of the KD value of the antigen-binding molecule for C1s in the acidic pH range to the KD value of the antigen-binding molecule for C1s in the neutral pH range, compared to that of a first reference antibody comprising two heavy chain variable regions and two light chain variable regions. In this embodiment, the heavy chain variable region of the first reference antibody comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region of the first reference antibody comprises the amino acid sequence of SEQ ID NO: 13.

[0060] 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 positions according to the Kabat numbering system: 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 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.

[0061] 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 positions according to the Kabat numbering system: 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 Light chain: L25, L28, L91, L92, L94, L95, L96 and L97.

[0062] 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 positions selected from the following positions according to the Kabat numbering system: 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 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.

[0063] In some embodiments, any one or more amino acids in the heavy chain variable region and / or light chain variable region are substituted with a histidine at the following positions according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96.

[0064] In some embodiments, the heavy chain variable region and / or the light chain variable region comprises one, two, three, four, or five histidines substituted for amino acid residues at the following positions according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96.

[0065] In some embodiments, the heavy chain variable region and / or the light chain variable region comprises at least one histidine substituted residue at one or more of the following positions and CDR or FR amino acid positions according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96.

[0066] In some embodiments, the heavy chain variable region and / or the light chain variable region comprises at least one histidine substituted residue at the following positions according to the Kabat numbering system: 1) L92 and L94 2) L92 and L95 3) L94 and L95 4) L92, L94, and L95 5) H65 and L92 6) H65 and L94 7) H65 and L95 8) H65, L92, and L94 9) H65, L92, and L95 10) H65, L94, and L95 11) H65, L92, L94, and L95 12) H99 and L92 13) H99 and L94 14) H99 and L95 15) H99, L92, and L94 16) H99, L92, and L95 17) H99, L94, and L95 18) H99, L92, L94, and L95 19) H65 and H99 20) H65, H99, and L92 21) H65, H99, and L94 22) H65, H99, and L95 23) H65, H99, L92, and L94 24) H65, H99, L92, and L95 25) H65, H99, L94, and L95 26) H65, H99, L92, L94, and L95, or 27)H27, H99, and L95.

[0067] In this embodiment, the variable region preferably comprises at least one of the following amino acids: a histidine at position 27 in the heavy chain variable region, a proline at position 59 in the heavy chain variable region, and a histidine at position 96 in the light chain variable region (all numbers according to the Kabat numbering system).

[0068] In another embodiment, the heavy chain variable region and / or the light chain variable region may contain at least one amino acid other than histidine that can increase the KD(acidic pH range) / KD(neutral pH range), which is the ratio of the KD value of the antigen-binding molecule for C1s in the acidic pH range to the KD value of the antigen-binding molecule for C1s in the neutral pH range, compared to that of a first reference antibody comprising two heavy chain variable regions and two light chain variable regions. In this embodiment, the heavy chain variable region of the first reference antibody comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region of the first reference antibody comprises the amino acid sequence of SEQ ID NO: 13.

[0069] In this embodiment, the heavy chain variable region and / or the light chain variable region may comprise the following amino acids at one or more of the following positions according to the Kabat numbering system: Heavy chain: D26, I26, T26, L26, V26, S26, S27, L27, Y27, I27, N28, A28, E28, G28, S28, D28, I29, G30, K30, R30, Y30, E30, D30, Q30, W30, I30, D31, E31, D32, L34, M34, R34, Q35, L35, I35, M49, Q51, G52, E53, P54, K57, A59, G60, P60, G61, E62, R64, E65, A97, E98, and T102; and Light chain: E24, E28, T28, F30, M31, K31, A33, E33, P34, G34, A50, T50, D51, G53, A56, L93, Y93, E95, K95, E97, G97, N97 and D97.

[0070] In one embodiment, the heavy chain variable region and / or the light chain variable region contains at least one amino acid that can enhance the binding ability of the antigen-binding molecule to its antigen in the neutral pH range compared to that of the first reference antibody.

[0071] In this embodiment, the heavy chain variable region and / or the light chain variable region may comprise the following amino acids at one or more of the following positions according to the Kabat numbering system: Heavy chain: Y27, I27, S28, D28, K30, R30, Y30, E30, D30, Q30, H30, W30, I30, D31, E31, D32, L34, M34, Q35, L35, M49, M51, E51, H51, I53, P53, S54, A59, P60, G61, R64, V96, Q96, W100a, N100b, G101, I102 and V102; and Light chain: S24, P24, E25, V26, P26, E26, D27, Y27, Q28, K28, M29, Y29, W29, E53, G53, Q54, E54, Y54, E56, A56, G56, V56, and F92.

[0072] In one embodiment, the Fc region comprises at least one amino acid that can enhance the binding ability of the antigen-binding molecule to FcRn in the acidic pH range compared to that of a second reference antibody comprising the paired Fc region. In this embodiment, the Fc region of the second reference antibody comprises the amino acid sequence of SEQ ID NO: 14.

[0073] In this embodiment, the Fc region comprises: (a) Ala at position 434; Glu, Arg, Ser, or Lys at position 438; and Glu, Asp, or Gln at position 440 according to the EU numbering system; (b) Ala at position 434; Arg or Lys at position 438; and Glu or Asp at position 440 according to the EU numbering system; (c) 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, according to the EU numbering system; (d) 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, according to the EU numbering system. (e) 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 according to the EU numbering system; or (f) 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, according to EU numbering.

[0074] WO2013 / 046704 specifically reports double amino acid residue substitutions of Q438R / S440E, Q438R / S440D, Q438K / S440E, and Q438K / S440D according to EU numbering, which, when combined with amino acid substitutions that may enhance binding to FcRn under acidic conditions, result in significantly reduced binding to rheumatoid factor.

[0075] In this embodiment, the Fc region preferably comprises a combination of amino acid substitutions selected from the group consisting of: (I)EUナンバリングによる、(a)N434A / Q438R / S440E;(b)N434A / Q438R / S440D;(c)N434A / Q438K / S440E;(d)N434A / Q438K / S440D;(e)N434A / Y436T / Q438R / S440E;(f)N434A / Y436T / Q438 R / S440D;(g)N434A / Y436T / Q438K / S440E;(h)N434A / Y436T / Q438K / S440D;(i)N434A / Y4 36V / Q438R / S440E;(j)N434A / Y436V / Q438R / S440D;(k)N434A / Y436V / Q438K / S440E;(l) N434A / Y436V / Q438K / S440D;(m)N434A / R435H / F436T / Q438R / S440E;(n)N434A / R435H / F436T / Q438R / S440D;(o)N434A / R435H / F436T / Q438K / S440E;(p)N434A / R435H / F436T / Q 438K / S440D;(q)N434A / R435H / F436V / Q438R / S440E;(r)N434A / R435H / F436V / Q438R / S4 40D;(s)N434A / R435H / F436V / Q438K / S440E;(t)N434A / R435H / F436V / Q438K / S440D;(u) M428L / N434A / Q438R / S440E;(v)M428L / N434A / Q438R / S440D;(w)M428L / N434A / Q438K / S440E;(x)M428L / N434A / Q438K / S440D;(y)M428L / N434A / Y436T / Q438R / S440E;(z)M428L / N434A / Y436T / Q438R / S440D;(aa)M428L / N434A / Y436T / Q438K / S440E;(ab)M428L / N434A / Y436T / Q438K / S440D;(ac)M428L / N434A / Y436V / Q438R / S440E;(ad)M428 L / N434A / Y436V / Q438R / S440D;(ae)M428L / N434A / Y436V / Q438K / S440E;(af)M428L / N434A / Y436V / Q438K / S440D;or; (II) According to EU numbering, (a) N434A / Q438R / S440E; (b) N434A / Y436T / Q438R / S440E; (c) N434A / Y436V / Q438R / S440E; (d) M428L / N434A / Q438R / S440E; (e) M428L / N434A / Y436T / Q438R / S440E ;(f)M428L / N434A / Y436V / Q438R / S440E;(g)L235R / G236R / S239K / M428L / N434A / Y436T / Q4 38R / S440E; and (h)L235R / G236R / A327G / A330S / P331S / M428L / N434A / Y436T / Q438R / S440E.

[0076] 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 numbers 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 numbers according to the EU numbering system).

[0077] In one embodiment, the Fc region comprises at least one amino acid that can enhance the binding ability of the antigen-binding molecule to an Fcγ receptor in the neutral pH region compared to a second reference antibody.

[0078] In this embodiment, the Fc region preferably comprises at least one or more amino acids selected from the group consisting of: In the Fc region according to EU numbering, either Lys or Tyr at amino acid position 221; any one of Phe, Trp, Glu, and Tyr at amino acid position 222; any one of Phe, Trp, Glu, and Lys at amino acid position 223; any one of Phe, Trp, Glu, and Tyr at amino acid position 224; any one of Glu, Lys, and Trp at amino acid position 225; any one of Glu, Gly, Lys, and Tyr at amino acid position 227; any one of Glu, Gly, Lys, and Tyr at amino acid position 228; any one of Ala, Glu, Gly, and Tyr at amino acid position 230; any one of Glu, Gly, Lys, Pro, and Tyr at amino acid position 231; any one of Glu, Gly, Lys, and Tyr at amino acid position 232; 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; 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; 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; 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; 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; 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; 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; any one of Ala, Ile, Met, and Thr at amino acid position 240; any one of Asp, Glu, Leu, Arg, Trp, and Tyr at amino acid position 241; any one of Leu, Glu, Leu, Gln, Arg, Trp, and Tyr at amino acid position 243; His at amino acid position 244; Ala at amino acid position 245; any one of Asp, Glu, His, and Tyr at amino acid position 246; any one of Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, and Tyr at amino acid position 247; any one of Glu, His, Gln, and Tyr at amino acid position 249; Either Glu or Gln at amino acid position 250; Phe at amino acid position 251; any one of Phe, Met, and Tyr at amino acid position 254; any one of Glu, Leu, and Tyr at amino acid position 255; any one of Ala, Met, and Pro at amino acid position 256; any one of Asp, Glu, His, Ser, and Tyr at amino acid position 258; any one of Asp, Glu, His, and Tyr at amino acid position 260; any one of Ala, Glu, Phe, Ile, and Thr at amino acid position 262; any one of Ala, Ile, Met, and Thr at amino acid position 263; 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; 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; any one of Ala, Ile, Met, and Thr at amino acid position 266; 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; any one of Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, and Trp at amino acid position 268; any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 269; any one of Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, and Tyr at amino acid position 270; 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; any one of Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 272; either Phe or Ile at amino acid position 273; 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; either Leu or Trp at amino acid position 275; any one of Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 276; 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; Ala at amino acid position 279; any one of Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, and Tyr at amino acid position 280; any one of Asp, Lys, Pro, and Tyr at amino acid position 281; any one of Glu, Gly, Lys, Pro, and Tyr at amino acid position 282; any one of Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, and Tyr at amino acid position 283; any one of Asp, Glu, Leu, Asn, Thr, and Tyr at amino acid position 284; any one of Asp, Glu, Lys, Gln, Trp, and Tyr at amino acid position 285; any one of Glu, Gly, Pro, and Tyr at amino acid position 286; any one of Asn, Asp, Glu, and Tyr at amino acid position 288; any one of Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, and Tyr at amino acid position 290; any one of Asp, Glu, Gly, His, Ile, Gln, and Thr at amino acid position 291; any one of Ala, Asp, Glu, Pro, Thr, and Tyr at amino acid position 292; any one of Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 293; any one of Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, and Tyr at amino acid position 294; 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; any one of Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, and Val at amino acid position 296; 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; any one of Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 298; 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; 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; any one of Asp, Glu, His, and Tyr at amino acid position 301; Ile at amino acid position 302; any one of Asp, Gly, and Tyr at amino acid position 303; any one of Asp, His, Leu, Asn, and Thr at amino acid position 304; any one of Glu, Ile, Thr, and Tyr at amino acid position 305; any one of Ala, Asp, Asn, Thr, Val, and Tyr at amino acid position 311; Phe at amino acid position 313; Leu at amino acid position 315; either Glu or Gln at amino acid position 317; any one of His, Leu, Asn, Pro, Gln, Arg, Thr, Val, and Tyr at amino acid position 318; any one of Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, and Tyr at amino acid position 320; any one of Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr, Val, Trp, and Tyr at amino acid position 322; Ile at amino acid position 323; any one of Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, and Tyr at amino acid position 324; 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; any one of Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, and Tyr at amino acid position 326; 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; 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; 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; 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; any one of Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, and Tyr at amino acid position 331; 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; any one of Ala, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, and Tyr at amino acid position 333; any one of Ala, Glu, Phe, Ile, Leu, Pro, and Thr at amino acid position 334; any one of Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, and Tyr at amino acid position 335; any one of Glu, Lys, and Tyr at amino acid position 336; any one of Glu, His, and Asn at amino acid position 337; any one of Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, and Thr at amino acid position 339; either Ala or Val at amino acid position 376; either Gly or Lys at amino acid position 377; Asp at amino acid position 378; Asn at amino acid position 379; any one of Ala, Asn, and Ser at amino acid position 380; either Ala or Ile at amino acid position 382; Glu at amino acid position 385; Thr at amino acid position 392; Leu at amino acid position 396; Lys at amino acid position 421; Asn at amino acid position 427; either Phe or Leu at amino acid position 428; Met at amino acid position 429; Trp at amino acid position 434; Ile at amino acid position 436; and any one of Gly, His, Ile, Leu, and Tyr at amino acid position 440;

[0079] In this embodiment, the Fc region is more preferably In the Fc region according to EU numbering, Asp at amino acid position 238, and Glu at amino acid position 328 The present invention includes at least one or more of the following:

[0080] 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 numbers according to the EU numbering system). The Fc region further preferably 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 according to the EU numbering system).

[0081] In one embodiment, the isoelectric point (pI) of the antigen-binding molecule is increased by modification of the Fc region. In this embodiment, the antigen-binding molecule with increased pI comprises at least two amino acid modifications in the Fc region compared to its parent Fc region. In a further embodiment, each amino acid modification increases the isoelectric point (pI) of the Fc region compared to its parent Fc region. In a further embodiment, the amino acid may be exposed on the surface of the region. In a further embodiment, the antigen-binding molecule comprises an Fc region and an antigen-binding domain. In a further embodiment, the antigen-binding activity of the antigen-binding domain is altered by ionic concentration conditions.

[0082] In a further embodiment, the pi increased Fc region of the present invention comprises at least two amino acid modifications 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 pi increased Fc region comprises Arg or Lys at each of the selected positions.

[0083] In certain embodiments, the Fc region with increased pi comprises an arginine at position 311 and an arginine at position 343 (both numbers according to the EU numbering system).

[0084] In one embodiment, the isoelectric point (pI) of the antigen-binding molecule is reduced by modification of the heavy chain variable region and / or the light chain variable region. In this embodiment, the antigen-binding molecule with reduced pI contains at least one amino acid modification in the heavy chain variable region and / or the light chain variable region compared to its parent region. The reduction in pI by modification of the heavy chain variable region and / or the light chain variable region can contribute to improving the PK of the antigen-binding molecule.

[0085] In this embodiment, the heavy chain variable region and / or the light chain variable region may comprise the following amino acids at one or more of the following positions according to the Kabat numbering system: Heavy chain: D26, E28, D28, E30, D30, D31, E31, D32, E51, E53, E62 and E65; and Light chain: E24, E25, E26, E28, E33, D51, E53, E54, E56, E95, E97 and D97.

[0086] In one embodiment, the heavy chain variable region and / or the light chain variable region comprises at least one amino acid that can improve the stability of the antigen-binding molecule compared to that of the first reference antibody.

[0087] In this embodiment, the heavy chain variable region and / or the light chain variable region may comprise the following amino acids at one or more of the following positions according to the Kabat numbering system: Heavy chain: G52, A96, V96, Q96, E98 and G101; and Light chain: E28, Q28, K28, T28, E53 and G53.

[0088] In this embodiment, either or both of alanine at position 96 in the heavy chain variable region and glutamic acid at position 53 in the light chain variable region (both numbers according to the Kabat numbering system) are preferred as the above amino acid.

[0089] In one embodiment, the antigen-binding molecule comprises a constant region. In this embodiment, the Fc region is present in the constant region. In this embodiment, the constant region preferably comprises at least one amino acid that can reduce the immunogenicity of the antigen-binding molecule. In this embodiment, arginine at position 214 according to the EU numbering system is preferred as such an amino acid.

[0090] In one embodiment, the Fc region of the antigen-binding molecule contains at least one amino acid that can reduce the binding activity to rheumatoid factor. In this embodiment, such amino acids are preferably either or both of arginine at position 438 and glutamic acid at position 440 (both numbers according to the EU numbering system).

[0091] In one aspect, the antigen-binding molecule binds to C1s, and the binding of the antigen-binding molecule to C1s competes with an 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. In this aspect, the antigen-binding molecule may have the heavy chain variable region, light chain variable region, and Fc region of any one of the above embodiments.

[0092] In one aspect, the antigen-binding molecule binds to C1s and comprises either or both of a VH region comprising 95% identity to the amino acid sequence of SEQ ID NO: 18 and a VL region comprising 95% identity to the amino acid sequence of SEQ ID NO: 19. In this aspect, the antigen-binding molecule may have the heavy chain variable region, the light chain variable region, and the Fc region of any one of the above embodiments.

[0093] In one aspect, the antigen-binding molecule binds to C1s and comprises either or both of a heavy chain variable region comprising HVR-H1 of SEQ ID NO: 20, HVR-H2 of SEQ ID NO: 21, or 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, or 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 of any one of the above embodiments.

[0094] In one aspect, the antigen binding molecule binds to C1s and does not compete with the binding of C1q to C1s. In this aspect, the antigen binding molecule may have a heavy chain variable region, a light chain variable region, and an Fc region according to any one of the above embodiments.

[0095] In one aspect, the antigen binding molecule binds to C1s and its epitope is within the CCP-SP domain of C1s. In this aspect, the antigen binding molecule can have a heavy chain variable region, a light chain variable region, and an Fc region in any one of the above embodiments.

[0096] In one aspect, the antigen-binding molecule can bind to C1s and reduce the plasma C1q concentration in an individual to 20% or less of the individual's baseline. In this aspect, the antigen-binding molecule can have the heavy chain variable region, light chain variable region, and Fc region of any one 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 of the antigen-binding molecule can be adjusted to be in excess of physiological plasma cynomolgus monkey C1s and C1q concentrations immediately after injection. Blood can be collected 1 day before injection and 5 minutes, 2, 8 hours, 1, 2, 4, 7, 14, 21, and 28 days after injection. The blood samples can be immediately centrifuged to separate the plasma samples. Cynomolgus monkey C1s and C1q plasma concentrations can be measured by LC / ESI-MS / MS at each sample collection time point.

[0097] III. Compositions and Methods In one aspect, the antigen binding molecules of the present invention are useful, for example, in the diagnosis or treatment of a complement-mediated disease or disorder.

[0098] In one embodiment, an anti-C1s antibody is exemplified as the above antigen-binding molecule. In this specification, the anti-C1s antibody described below can be replaced with an antigen-binding molecule that binds to C1s.

[0099] A. Exemplary Anti-C1s Antibodies 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, and whose binding activity varies depending on ion concentration. In a particular embodiment, the binding activity of the anti-C1s antibody varies depending on pH, i.e., hydrogen ion (proton) concentration. In a particular embodiment, the binding activity of the anti-C1s antibody varies depending on calcium concentration. In a particular embodiment, the binding activity of the anti-C1s antibody varies depending on both pH and calcium concentration. Such an antibody is expected to be particularly excellent as a pharmaceutical, since it can reduce the dose and frequency of administration in patients, and therefore the total dose can be reduced.

[0100] In one aspect, when the isolated anti-C1s antibody of the present invention is measured under high calcium concentrations at both neutral and acidic pH, 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 more. In one aspect, when the isolated anti-C1s antibody of the present invention is measured under high calcium concentrations at both neutral and acidic pH, 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 more. In one aspect, when the isolated anti-C1s antibody of the present invention is measured under high calcium concentrations at neutral pH and low calcium concentrations at acidic pH, 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. In some embodiments, for an isolated anti-C1s antibody of the present invention, when measured under low calcium concentrations at both neutral and acidic pH, 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 more, wherein the anti-C1s antibody binds to C1s in a dimeric state. In some embodiments, for an isolated anti-C1s antibody of the present invention, when measured under low calcium concentrations at both neutral and acidic pH, 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 more, wherein the anti-C1s antibody binds to C1s in a dimeric state.

[0101] Without being bound to a particular theory, if 1) the epitope structure of C1s bound by the antibody of the present invention may conformationally change in the absence of calcium, thereby changing the affinity of the antibody, or 2) the interaction (affinity type or avidity type) of the antibody of the present invention may change depending on the state of C1s (monomer state or dimer state), measurements using specific conditions (under high calcium concentration at neutral pH and low calcium concentration at acidic pH) may be used to evaluate the ratio of KD values ​​(KD(acidic pH) / KD(neutral pH)).

[0102] In other words, the antibodies of the invention bind to C1s with higher affinity at neutral pH than at acidic pH as described below in (i) or (iii): (i) When measured at high calcium concentrations at both neutral and acidic pH, 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)) is 2 or more; (ii) when measured under high calcium concentrations at both neutral and acidic pH, 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 more; (iii) When measured under high calcium concentrations at neutral pH and under low calcium concentrations at acidic pH, 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)) is 5 or greater.

[0103] More generally, without being bound by any particular theory, if 1) the epitope structure of a particular antigen bound by an antibody of the present invention may be conformationally altered in the absence of calcium, thereby altering the affinity of the antibody, or 2) the interaction (affinity or avidity type) of an antibody of the present invention may vary depending on the state of the antigen (monomeric or dimeric state), measurements using specific conditions (high calcium concentration at neutral pH and low calcium concentration at acidic pH) may be used to evaluate 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 mentioned below, KD can be expressed as k off / k on It is defined as the ratio of k between acidic and neutral conditions. off The ratio of values, ie, (koff(acidic pH) / koff(neutral pH)), can also be used for comparison between affinity at acidic pH and affinity at neutral pH.

[0104] Thus, the antibodies of the present invention bind to antigens with higher affinity at neutral pH than at acidic pH as follows: the ratio of the KD value of antigen-binding activity at acidic pH to the KD value of antigen-binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) is 5 or greater when measured under high calcium concentrations at neutral pH and under low calcium concentrations at acidic pH.

[0105] In one aspect, for an isolated anti-C1s antibody of the present invention, when measured under high calcium concentrations at both neutral and acidic pH, 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 more.

[0106] In one aspect, for an isolated anti-C1s antibody of the present invention, when measured under high calcium concentration at neutral pH and under low calcium concentration at acidic pH, 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.

[0107] In one aspect, for an isolated anti-C1s antibody of the present invention, when measured under low calcium concentrations at both neutral and acidic pH, 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.

[0108] The above KD ratio, i.e., KD(acidic pH) / KD(neutral pH), can be compared between a parent antibody (i.e., the original antibody before the 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 into the original (parent) antibody. The original (parent) antibody can be any known antibody or a newly isolated antibody, so long as it specifically binds to C1s. Thus, in one aspect, for an isolated anti-C1s antibody of the present invention, 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)) 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 C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)) of its original (parent) antibody. In other words, the present invention provides an isolated anti-C1s antibody in which one or more amino acid mutations (e.g., addition, insertion, deletion or substitution) have been introduced into a parent (original) antibody, and the ratio of (i) to (ii) as follows 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 C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH of the isolated anti-C1s antibody (KD(acidic pH) / KD(neutral pH)); (ii) the ratio of the KD value of C1s binding activity at acidic pH to the KD value of C1s binding activity at neutral pH of the parent (original) antibody (KD(acidic pH) / KD(neutral pH)). These KD ratios can be measured under any (high or low) calcium concentration, for example, under high calcium concentrations at both neutral and acidic pH, or under high calcium concentrations at neutral pH and low calcium concentrations at acidic pH. In a further aspect, the dissociation rate constant (kd) can be used instead of the KD to assess pH and / or Ca dependence.

[0109] In one aspect, the antibody of the present invention has different antigen-binding activity between intracellular 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 characteristic of the environment in an endosome, and "extracellular conditions" preferably refer to an environment characteristic of the environment in plasma. An antibody having the property of having an antigen-binding activity that varies depending on an ion concentration can be obtained by screening a large number of antibodies for domains having such a property. For example, an antibody having the above property can be obtained by producing a large number of antibodies whose sequences are different from each other by the hybridoma method or the antibody library method, and measuring their antigen-binding activity under different ion concentrations. The B cell cloning method is one example of a method for screening such an antibody. Furthermore, as described below, at least one characteristic amino acid residue that can confer to an antibody the property of having an antigen-binding activity that varies depending on ion concentration is identified, and a library of multiple antibodies having different sequences while sharing the characteristic amino acid residue as a common structure is prepared. Such a library can be screened to effectively isolate antibodies having the above-mentioned properties.

[0110] In one aspect, the present invention provides an antibody that binds to C1s with higher affinity at neutral pH than at acidic pH. In another aspect, the present invention provides an anti-C1s antibody that exhibits pH-dependent binding to C1s. As used herein, the expression "pH-dependent binding" means "reduced binding at acidic pH compared to binding at neutral pH", and both expressions may be interchangeable. For example, an anti-C1s antibody "having pH-dependent binding properties" includes an antibody that binds to C1s with higher affinity at neutral pH than at acidic pH.

[0111] In certain embodiments, when measured under high calcium concentrations at both neutral and acidic pH, 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)) is equal to or greater than 2. In certain embodiments, the antibodies of the present invention bind to C1s with 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 affinity at neutral pH than at acidic pH.

[0112] In certain embodiments, when measured under high calcium concentrations at both neutral and acidic pH, 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 greater than or equal to 2. In certain embodiments, the antibodies of the present invention bind to C1s with 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 affinity at neutral pH than at acidic pH.

[0113] In a particular aspect, when measured under high calcium concentration at neutral pH and under low calcium concentration at acidic pH, 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)) is 2 or greater. In certain embodiments, antibodies of the invention bind to C1s with 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, 200, 400, 1000, 10000, or more fold greater affinity at neutral pH than at acidic pH.

[0114] In the above example, 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 later in this specification. In some embodiments, KD(acidic pH) / KD(neutral pH), for example KD(pH 5.8) / KD(pH 7.4), can be 2 to 10,000. In the above example, for example, the acidic pH is 5.8, and the neutral pH is 7.4, so koff(acidic pH) / koff(neutral pH) is koff(pH 5.8) / koff(pH 7.4). In this regard, examples of acidic pH and neutral pH are described in detail later in this specification. In some embodiments, the koff(acidic pH) / koff(neutral pH), for example koff(pH 5.8) / koff(pH 7.4), can be between 2 and 10,000.

[0115] When the antigen is a soluble protein, the binding of the antibody to the antigen can result in an increased plasma half-life of the antigen (i.e., reduced clearance of the antigen from plasma) because the antibody can have a longer half-life in plasma than the antigen itself and can function as a carrier of the antigen. This is due to the recycling of the antigen-antibody complex by FcRn via the endosomal pathway in cells (Roopenian and Akilesh (2007) Nat Rev Immunol 7(9): 715-725). However, antibodies with pH-dependent binding properties, which bind antigens in the neutral extracellular environment but release antigens into acidic endosomal compartments after intracellular entry, are expected to have superior properties in terms of antigen neutralization and clearance compared to their 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).

[0116] In one aspect, the present invention provides antibodies that bind to C1s with higher affinity under high calcium concentration conditions than under low calcium concentration conditions.

[0117] In the present invention, preferred metal ions include, for example, calcium ions. Calcium ions are involved in the regulation of many biological phenomena, including muscle contraction, such as skeletal muscle, smooth muscle and cardiac muscle; activation of leukocytes, such as motility and phagocytosis; activation of platelets, such as shape change and secretion; activation of lymphocytes; activation of mast cells, including secretion of histamine; cell responses mediated by catecholamine alpha receptors or acetylcholine receptors; exocytosis; release of transmitters from neuronal terminals; and axonal 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 thought to be derived from a common origin in terms of molecular evolution. In addition, many calcium binding motifs are known. Such well-known motifs include, for example, cadherin domains, the EF hands of calmodulin, the C2 domain of protein kinase C, the Gla domain of the blood clotting protein factor IX, the C-type lectins of the asialoglycoprotein receptor and the mannose-binding receptor, the A domain of the LDL receptor, annexins, the thrombospondin type 3 domain and the EGF-like domain.

[0118] In the present invention, when the metal ion is a calcium ion, it is desirable that the antigen-binding activity under a low calcium ion concentration condition is lower than that under a high calcium ion concentration condition. On the other hand, the intracellular calcium ion concentration is lower than the extracellular calcium ion concentration. Conversely, 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 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 in early endosomes in vivo. On the other hand, 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 0.5 mM to 2.5 mM, which is close to the calcium ion concentration in plasma (blood). In the present invention, it is preferable that the low calcium ion concentration is the calcium ion concentration in endosomes, and the high calcium ion concentration is the calcium ion concentration in plasma. When comparing the antigen-binding activity level under low calcium ion concentration and high calcium ion concentration, the binding of the antibody of the present invention is preferably stronger under high calcium ion concentration than under low calcium ion concentration. In other words, the antigen-binding activity of the antibody of the present invention is preferably lower under low calcium ion concentration than under high calcium ion concentration. When the level of binding activity is expressed as a dissociation constant (KD), the value of KD (low calcium ion concentration) / KD (high calcium ion concentration) is greater than 1, preferably 2 or more, more preferably 10 or more, and even more preferably 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 400, 1000, 10000, or more. The upper limit of the value of KD (low calcium ion concentration) / KD (high calcium ion concentration) is not particularly limited, and may be any value such as 100, 400, 1000, or 10000, as long as it can be produced by the technology of a person skilled in the art. It is also possible to use the dissociation rate constant (kd) instead of KD. If it is difficult to calculate the KD value, the activity may be evaluated based on the level of binding reactivity when the analyte is run at the same concentration in the Biacore.When an antigen is flowed over a chip on which an antigen-binding molecule of the present invention is immobilized, the binding reactivity under a low calcium concentration is preferably 1 / 2 or less, more preferably 1 / 3 or less, even more preferably 1 / 5 or less, and particularly preferably 1 / 10 or less, of the binding reactivity under a high calcium concentration. In general, it is known that the calcium ion concentration outside a cell (e.g., in plasma) in a living body is high, and the calcium ion concentration inside the cell (e.g., in an endosome) is low. Therefore, in the present invention, it is preferable that the extracellular condition is a high calcium ion concentration and the intracellular condition is a low calcium ion concentration. When the antigen-binding molecule (e.g., an antibody) of the present invention is given a property that the antigen-binding activity is lower under intracellular calcium ion concentration conditions than under extracellular calcium ion concentration conditions, the antigen bound to the antigen-binding molecule of the present invention outside the cell dissociates from the antigen-binding molecule of the present invention inside the cell, thereby enhancing the uptake of the antigen from outside the cell into the cell. When such an antibody is administered to a living body, it is possible to reduce the antigen concentration in plasma and reduce the physiological activity of the antigen in the living body, and therefore the antibody of the present invention is useful. Methods for screening antigen-binding domains or antibodies with lower antigen-binding activity under low calcium ion concentration conditions compared to high calcium ion concentration conditions include, for example, the methods described in WO2012 / 073992 (e.g., paragraphs 0200 to 0213). The method for imparting the property of weaker antigen-binding activity under low calcium ion concentration conditions compared to high calcium ion concentration conditions to the antigen-binding domain of the present invention is not particularly limited and may be performed by any method. Specifically, such methods are described in Japanese Patent Application No. 2011-218006, and include, for example, a method of substituting at least one amino acid residue in the antigen-binding domain with an amino acid residue having metal chelating activity, and / or a method of inserting at least one amino acid residue having metal chelating activity into the antigen-binding domain. An antigen-binding molecule of the present invention in which at least one amino acid residue in the antigen-binding domain is substituted with an amino acid residue having metal chelating activity and / or at least one amino acid residue having metal chelating activity is inserted into the antigen-binding domain is one of the preferred embodiments of the antigen-binding molecule of the present invention.

[0119] The amino acid residues having metal chelating activity preferably include, for example, serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid.Furthermore, the amino acid residues that change the antigen-binding activity of the antigen-binding domain depending on calcium ion concentration preferably include, for example, amino acid residues that form a calcium-binding motif. Calcium-binding motifs are well known to those skilled in the art and have been reported 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-hands of troponin C, calmodulin, parvalbumin, and myosin light chain; the C2 domain of protein kinase C; the Gla domain of blood clotting protein factor IX; the C-type lectins of asialoglycoprotein receptor and mannose-binding receptor, ASGPR, CD23, and DC-SIGN; the A-domain of the LDL receptor; the annexin domain; the cadherin domain; the thrombospondin type 3 domain; and the EGF-like domain are preferably used as calcium-binding motifs.

[0120] The antigen-binding domain of the present invention may contain amino acid residues that change the antigen-binding activity depending on the calcium ion concentration, such as the amino acid residues having metal chelating activity and the amino acid residues that form a calcium-binding motif. The position of such amino acid residues in the antigen-binding domain is not particularly limited, and may be any position as long as the antigen-binding activity changes depending on the calcium ion concentration. In addition, such amino acid residues may be contained alone or in combination of two or more as long as the antigen-binding activity changes depending on the calcium ion concentration. Suitable examples of such amino acid residues include serine, threonine, asparagine, glutamine, aspartic acid, and glutamic acid. When the antigen-binding domain is a variable region of an antibody, the heavy chain variable region and / or the light chain variable region may contain such amino acid residues. In a preferred embodiment, such amino acid residues may be contained in the CDR3 of the heavy chain variable region, and more preferably, may be contained at positions 95, 96, 100a, and / or 101 of the CDR3 of the heavy chain variable region according to the Kabat numbering.

[0121] In another preferred embodiment, those amino acid residues may be contained in CDR1 of the light chain variable region, more preferably in position 30, 31 and / or 32 of the CDR1 of the light chain variable region as defined by Kabat numbering. In yet another preferred embodiment, those amino acid residues may be contained in CDR2 of the light chain variable region, more preferably in position 50 of the CDR2 of the light chain variable region as defined by Kabat numbering. In yet another preferred embodiment, those amino acid residues may be contained in CDR3 of the light chain variable region, more preferably in position 92 of the CDR3 of the light chain variable region as defined by Kabat numbering.

[0122] Furthermore, the above aspects 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, and more preferably, the amino acid residue may be contained in one or more of positions 30, 31, 32, 50 and / or 92 as defined by the Kabat numbering in the light chain variable region.

[0123] By preparing a library of numerous antigen-binding domains that have different sequences but share a common structure of amino acid residues that change their antigen-binding activity in response to calcium ion concentration as described above, and screening from such a library, it is possible to efficiently obtain antigen-binding domains that have binding activity to a desired antigen and whose antigen-binding activity changes in response to calcium ion concentration.

[0124] The "affinity" of an antibody to C1s is, for the purposes of this disclosure, expressed by the KD of the antibody. The KD of an antibody refers to the equilibrium dissociation constant of the antibody-antigen interaction. The higher the KD value of an antibody's binding to its antigen, the weaker its binding affinity to that particular antigen. Thus, 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 acidic pH is higher than the KD of the antibody at neutral pH. For example, in the context of the present invention, an antibody is considered to bind C1s with higher affinity at neutral pH than at acidic pH if the KD of the antibody's binding to C1s at acidic pH is at least two times higher than the KD of the antibody's binding to C1s at neutral 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, or more times greater than the KD of the antibody binding to C1s at neutral pH. -7 M, 10-8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In another embodiment, the K value of the antibody at acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or even higher.

[0125] The binding properties for a particular antigen may also be expressed as the kd of an antibody. The kd of an antibody refers to the dissociation rate constant of an antibody for a particular antigen, and is expressed as the reciprocal of seconds (i.e., sec -1 The kd value is expressed in units of 1000 kd. An increased kd value means that the antibody binds weaker to its antigen. Thus, the present invention includes antibodies that bind to C1s with a higher kd value at acidic pH than at neutral pH. The present 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, or more times greater than the kd of the antibody's binding to C1s at neutral pH. In another embodiment, the kd value of the antibody at neutral pH is 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 In another embodiment, the kd value of the antibody at acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 It can be 1 / s, or even higher.

[0126] In certain instances, "reduced binding at acidic pH compared to binding at neutral pH" is expressed as the ratio of the antibody's KD value at acidic pH to the antibody's KD value at neutral pH (or vice versa). For example, if an antibody exhibits an acidic / neutral KD ratio of 2 or more, for the purposes of the present invention, the antibody may be considered to exhibit "reduced binding to C1s at acidic pH compared to binding to C1s at neutral pH". In certain exemplary embodiments, the acidic / neutral KD ratio of the antibody of the present invention may 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 times, or more. In another embodiment, the KD value of the antibody at neutral pH is 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 In another embodiment, the K value of the antibody at acidic pH is 10 -9 M, 10 -8 M, 10 -7 M, 10 -6 M, or even higher.

[0127] Alternatively, "reduced binding at acidic pH compared to binding at neutral pH" is expressed as the ratio of the antibody's koff value at acidic pH to the antibody's koff value at neutral pH (or vice versa). For example, if an antibody exhibits an acidic / neutral koff ratio of 2 or more, then for the purposes of the present invention, the antibody can be considered to exhibit "reduced binding to C1s at acidic pH compared to binding to C1s at neutral pH". In certain exemplary embodiments, the acidic / neutral koff ratio of the antibody of the present 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 times, or more.

[0128] In certain instances, "reduced binding at acidic pH compared to binding at neutral pH" is expressed as the ratio of the antibody's kd value at acidic pH to the antibody's kd value at neutral pH (or vice versa). For example, if an antibody exhibits an acidic / neutral kd ratio of 2 or greater, then for purposes of the present invention, the antibody may be considered to exhibit "reduced binding to C1s at acidic pH compared to binding to C1s at neutral pH". In certain exemplary embodiments, the acidic / neutral kd ratio for the antibodies of the present invention may 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 times, or more. In another embodiment, the kd value of the antibody at neutral pH is 10 -2 1 / s, 10 -3 1 / s, 10 -4 1 / s, 10 -5 1 / s, 10 -6 In another embodiment, the kd value of the antibody at acidic pH is 10 -3 1 / s, 10 -2 1 / s, 10 -1 It can be 1 / s, or even higher.

[0129] The expression "acidic pH" as used herein means a pH between 4.0 and 6.5. The expression "acidic pH" includes pH values ​​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 certain aspects, the "acidic pH" is 5.8 or 6.0.

[0130] As used herein, the expression "neutral pH" refers to a pH of 6.7 to about 10.0. The expression "neutral pH" 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 certain aspects, the "neutral pH" is 7.0 or 7.4.

[0131] The expression "under high calcium concentration conditions" or "high calcium concentration" as used herein 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, "high calcium concentration conditions" or "high calcium concentration" includes calcium concentration values ​​of 1.2 mM Ca 2+ Represents.

[0132] The expression "under low calcium concentration conditions" or "under low calcium concentration" as used herein 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 in early endosomes in vivo. The expression "under low calcium concentration conditions" or "at low calcium concentration" means 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 "under low calcium concentration" includes calcium concentration values ​​of 3.0 μM Ca 2+ Represents.

[0133] The KD and kd values ​​presented herein can be determined using a biosensor based on surface plasmon resonance to characterize antibody-antigen interaction. (See, for example, the Examples herein.) The KD and kd values ​​can be determined at 25 degrees Celsius (℃) or 37℃. This determination can be performed in the presence of 150 mM NaCl. In some embodiments, this determination can be performed using surface plasmon resonance technology, immobilizing the antibody and using the antigen as analyte, and using the following conditions: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate and 150 mM NaCl at 37 degrees Celsius (℃).

[0134] In one aspect, the present invention provides an anti-C1s antibody having pH dependence, comprising at least one histidine in a variable region, and wherein at least one amino acid in the variable region is 1) to reduce the non-specific binding activity of the antibody at acidic and / or neutral pH, or 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)) is increased. Antibodies are provided in which the amino acids have been substituted with other amino acids. In one aspect, the present invention provides an anti-C1s antibody having pH dependence, comprising at least one histidine in a variable region, and wherein at least one amino acid in the variable region is 1) to reduce the non-specific binding activity of the antibody at acidic and / or neutral pH, or 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)) is increased. The antibodies are provided in which the amino acid residues are substituted with amino acids selected from the group consisting of D, E, K, R and Q. In certain embodiments, the expression "non-specific binding activity" refers to the extracellular matrix (ECM) binding activity of an antibody. In certain aspects, the expression "non-specific binding activity" refers to the ECM binding activity of an antibody at acidic pH. 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 so as to reduce the ECM binding activity at acidic pH. 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 so as to reduce the ECM binding activity at neutral pH.

[0135] In one aspect, the present invention provides an anti-C1s antibody having pH dependency, in which at least one amino acid is substituted in the variable region such that the ECM binding activity of the antibody is reduced. In a particular embodiment, the ECM binding activity at acidic pH is reduced. In a particular embodiment, the ECM binding activity at neutral pH is reduced. In a particular embodiment, such an antibody refers to an antibody having one or more modifications in one or more hypervariable regions (HVRs) compared to a parent antibody that does not have such modifications, which modifications improve the ECM binding activity of the antibody to an antigen, i.e., reduce the ECM binding activity.

[0136] The method for measuring "binding to the extracellular matrix" is not particularly limited, and the measurement can be performed using an ELISA system that detects the binding between the polypeptide and the extracellular matrix by adding a polypeptide to a plate on which the extracellular matrix is ​​immobilized and adding a labeled antibody against the polypeptide. In particular, a measurement method using electrochemiluminescence (ECL) is preferred because it can detect the extracellular matrix binding ability with higher sensitivity. Specifically, a mixture of a polypeptide and a ruthenium antibody is added to a plate on which the extracellular matrix is ​​immobilized, and the binding between the polypeptide and the extracellular matrix can be measured using an ECL system that measures the electrochemiluminescence of ruthenium. The concentration of the polypeptide to be added can be set arbitrarily, but it is preferable to add it at a high concentration in order to increase the detection sensitivity of the extracellular matrix binding. The extracellular matrix used in the present invention may be derived from either plants or animals as long as it contains glycoproteins such as collagen, proteoglycan, fibronectin, laminin, entactin, fibrin, and perlecan, but in the present invention, an extracellular matrix derived from an animal is preferred, and for example, an extracellular matrix derived from an animal such as a human, mouse, rat, monkey, rabbit, or dog can be used. In particular, natural human extracellular matrix derived from humans is preferred for monitoring the improvement of pharmacokinetics in humans. Furthermore, the conditions for evaluating the binding of a polypeptide to an extracellular matrix are preferably in the neutral range (physiological conditions) of around pH 7.4, but do not necessarily have to be within the neutral range and may be evaluated in the acidic range (around pH 6.0). Furthermore, when evaluating the binding of a polypeptide to an extracellular matrix, an antigen molecule to which the polypeptide binds may be coexisted in order to evaluate the binding of a polypeptide / antigen molecule complex to the extracellular matrix.

[0137] In some embodiments, whether the non-specific binding activity of an antibody at acidic pH is reduced can be measured, for example, by using ELISA or ECL. In a further embodiment, for the isolated anti-C1s antibody of the present invention, the ECM binding value can be compared between the parent antibody (i.e., the original antibody before D, E, K, R and / or Q substitution) and an antibody in which one or more amino acid substitutions (D, E, K, R and / or Q) have been introduced into the original (parent) antibody (provided that the antibody contains at least one histidine in the variable region). The original (parent) antibody can be any known antibody or a newly isolated antibody, so long as it specifically binds to C1s. Thus, in one aspect, for the isolated anti-C1s antibody of the present invention, the ECM binding value of the substituted antibody is at least 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 8-fold, 10-fold lower than the ECM binding value of the original (parent) antibody.

[0138] Without being bound to a particular theory, the histidine residue of an antibody may interact with various residues around the histidine residue of the antibody. Such interactions may affect the structure of the antibody or the conformation of the CDR. Histidine is protonated and positively charged at acidic pH. Introducing a positively charged residue (e.g., arginine or lysine) into a position around histidine causes repulsion between the positively charged residue and the protonated histidine at acidic pH, thereby inducing a structural or conformational change in the antibody or CDR. Similarly, introducing a negatively charged residue (e.g., aspartic acid or glutamic acid) into a position around histidine causes an interaction between the negatively charged residue and the protonated histidine at acidic pH, thereby inducing a structural or conformational change in the antibody or CDR. These structural or conformational changes of the antibody or CDR that occur at acidic pH may affect the antigen binding of the antibody and reduce the binding affinity of the antibody to the antigen at acidic pH. In summary, the introduction of charged residues (e.g., arginine, lysine, aspartic acid, or glutamic acid) into positions surrounding the histidine residues of an antibody may reduce the binding affinity of the antibody for the antigen at acidic pH, thereby improving the pH dependence of the antibody-antigen interaction by a unique mechanism.

[0139] In one aspect, the present invention provides a method for increasing the ratio of the KD value of an antibody's antigen-binding activity at acidic pH to the KD value of the antigen-binding activity at neutral pH (KD(acidic pH) / KD(neutral pH)), comprising: 1) providing an antibody having pH dependency, the antibody containing at least one histidine in the variable region; 2) replacing at least one amino acid in a variable region of the antibody with an amino acid selected from the group consisting of D, E, K, R, Q and H; The present invention provides a method comprising: In one aspect, the invention provides a method of enhancing the clearance of an antigen from plasma, comprising: 1) providing an antibody having pH dependency, the antibody containing at least one histidine in the variable region; 2) replacing at least one amino acid in a variable region of the antibody with an amino acid selected from the group consisting of D, E, K, R, Q and H; The present invention provides a method comprising: In one aspect, the present invention provides a method for promoting antigen uptake into a cell via an antigen-binding molecule, comprising: 1) providing an antibody having pH dependency, the antibody containing at least one histidine in the variable region; 2) replacing at least one amino acid in a variable region of the antibody with an amino acid selected from the group consisting of D, E, K, R, Q and H; The present invention provides a method comprising: In one aspect, the present invention provides a method for increasing the number of antigens that can be bound by an antigen-binding molecule, comprising the steps of: 1) providing an antibody having pH dependency, the antibody containing at least one histidine in the variable region; 2) replacing at least one amino acid in a variable region of the antibody with an amino acid selected from the group consisting of D, E, K, R, Q and H; The present invention provides a method comprising: In one aspect, the present invention provides a method for enhancing the ability of an antigen-binding molecule to clear an antigen from plasma, comprising: 1) providing an antibody having pH dependency, the antibody containing at least one histidine in the variable region; 2) replacing at least one amino acid in a variable region of the antibody with an amino acid selected from the group consisting of D, E, K, R, Q and H; The present invention provides a method comprising:

[0140] In certain embodiments, in the above methods of the present invention, the distance between the histidine residue contained in the variable region and the substituted amino acid (i.e., D, E, K, R, Q or H) is less than 20 angstroms, less than 18 angstroms, less than 16 angstroms, less than 14 angstroms, less than 12 angstroms, less than 10 angstroms, less than 8 angstroms, less than 6 angstroms, less than 4 angstroms, or less than 2 angstroms.

[0141] In one aspect, the present invention provides a method for enhancing the clearance of C1s from plasma in an individual. In some embodiments, the method comprises administering to the individual an anti-C1s antibody of the present invention in an amount effective to enhance the clearance of C1s from plasma. The present invention also provides a method for enhancing the clearance of a complex of C1r and C1s from plasma in an individual. In some embodiments, the method comprises administering to the individual an anti-C1s antibody of the present invention in an amount effective to enhance the clearance of a complex of C1r and C1s from plasma. The present invention also provides a method for enhancing the clearance of a complex of C1q, C1r and C1s from plasma in an individual. In some embodiments, the method comprises administering to the individual an anti-C1s antibody of the present invention in an amount effective to enhance the clearance of a complex of C1q, C1r and C1s from plasma.

[0142] In another aspect, the present invention provides a method for removing C1s from plasma, the method comprising the steps of: (a) identifying an individual in need of removing C1s from his plasma; (b) providing an antibody that binds to C1s, the antibody binding to C1s through the antigen-binding (C1s-binding) domain of the antibody and having a KD(pH 5.8) / KD(pH 7.4) value of 2 to 10,000, where KD(pH 5.8) / KD(pH 7.4) is defined as the ratio of the KD for C1s at pH 5.8 to the KD for C1s at pH 7.4, where KD is determined using a surface plasmon resonance technique, the antibody binds to C1s in plasma in vivo and dissociates from the bound C1s under conditions present in endosomes in vivo, the antibody being a human IgG or a humanized IgG; and (c) administering the antibody to the individual. In a further aspect, such surface plasmon resonance techniques can be used at 37° C. and 150 mM NaCl. In a further aspect, such surface plasmon resonance techniques can be used with immobilized antibodies, antigens as analytes, and the following conditions: 10 mM MES buffer, 0.05% polyoxyethylenesorbitan monolaurate, and 150 mM NaCl at 37° C. In a further aspect, the dissociation rate constant (kd) can be used instead of the KD.

[0143] In another aspect, the present invention provides a method for removing C1s from plasma in a subject, the method comprising the steps of: (a) identifying a first antibody, the first antibody binding to C1s through an antigen-binding domain of the first antibody; (b) identifying a second antibody, the second antibody (1) binding to C1s through the antigen-binding (C1s-binding) domain of the second antibody, (2) having the same amino acid sequence as the first antibody except that at least one amino acid in the variable region of the first antibody has been substituted with histidine and / or at least one histidine has been inserted into the variable region of the first antibody, and (3) having a KD(pH 5.8) / KD(pH 7.4) value that is higher than the KD(pH 5.8) / KD(pH 7.4) value of the first antibody and is between 2 and 10,000, wherein KD(pH 5.8) / KD(pH 7.4) is defined as the ratio of KD for C1s at pH 5.8 to KD for C1s at pH 7.4, where KD is determined using surface plasmon resonance technology, (4) binds to C1s in plasma in vivo, (5) dissociates from bound C1s under conditions present in endosomes in vivo, and (6) is a human IgG or humanized IgG; (c) identifies a subject in need of reducing plasma C1s levels; and (d) administers a second antibody to the subject such that the subject's plasma C1s levels are reduced. In a further aspect, such surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In a further aspect, such surface plasmon resonance technology can be used at 37°C and 150 mM NaCl. In a further aspect, such surface plasmon resonance techniques can be used with immobilized antibodies, antigen as the analyte, and the following conditions: 10 mM MES buffer, 0.05% polyoxyethylenesorbitan monolaurate, and 150 mM NaCl at 37° C. In a further aspect, the dissociation rate constant (kd) can be used instead of the KD described above.

[0144] In another aspect, the present invention provides a method of removing C1s from plasma in a subject, comprising the steps of: (a) identifying a first antibody, the first antibody having an amino acid sequence identical to that of a second antibody that binds C1s through an antigen-binding domain of the first antibody, (2) at least one variable region of the first antibody has at least one more histidine residue than a corresponding variable region of the second antibody, and (3) has a KD(pH 5.8) / KD(pH 7.4) value that is higher than the KD(pH 5.8) / KD(pH 7.4) value of the second antibody and is between 2 and 10,000, wherein KD(pH 5.8) / KD(pH 7.4) is the ratio of the KD for C1s at pH 5.8 and pH 7.4 when the KD is determined using surface plasmon resonance technology. The present invention provides a method comprising the steps of: (a) determining a subject in need of reducing plasma C1s levels; and (b) administering the first antibody at least once to the subject such that the subject's plasma C1s level is reduced; (b) determining a subject in need of reducing plasma C1s levels; and (c) administering the first antibody at least once to the subject such that the subject's plasma C1s level is reduced. In a further aspect, such a surface plasmon resonance technique can be used at 37°C and 150 mM NaCl. In a further aspect, such a surface plasmon resonance technique can be used at 37°C and 150 mM NaCl. In a further aspect, such a surface plasmon resonance technique can be used with an antibody immobilized, an antigen as an analyte, and the following conditions: 10 mM MES buffer, 0.05% polyoxyethylene sorbitan monolaurate, and 150 mM NaCl at 37°C. The present invention also provides a method of inhibiting cleavage of complement component C4, wherein the antibody does not inhibit cleavage of complement component C2. In some instances, the antibody inhibits a component of the classical complement pathway, and in some instances, the component of the classical complement pathway is C1s. In a further aspect, the dissociation rate constant (kd) can be used in place of the KD.

[0145] In one aspect, the present disclosure provides a method of modulating 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 of modulating complement activation in an individual having a complement-mediated disease or disorder, 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, such a method inhibits complement activation. In some embodiments, the individual is a mammal. In some embodiments, the individual is a human. The administration can be by any route known to those skilled in the art, including those disclosed herein. In some embodiments, the administration is intravenous administration. In some embodiments, the administration is intrathecal administration.

[0146] In certain embodiments, the anti-C1s antibody of the present invention binds to C1s from two or more species. In certain embodiments, the anti-C1s antibody binds to C1s from humans and non-human animals. In certain embodiments, the anti-C1s antibody binds to C1s from humans, rats and monkeys (e.g., cynomolgus monkeys, rhesus monkeys, marmosets, chimpanzees and baboons).

[0147] In some embodiments, anti-C1s antibody variants are provided that 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.

[0148] In some embodiments, the anti-C1s antibodies of the invention comprise a histidine at one or more of the following positions according to the Kabat numbering system: 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 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, the anti-C1s antibodies of the invention comprise a histidine at one or more of the following positions according to the Kabat numbering system: 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 Light chain: L25, L28, L91, L92, L94, L95, L96 and L97.

[0150] In some embodiments, the anti-C1s antibody of the invention comprises at least one histidine substituted for one or more amino acid residues at positions selected from the following positions according to the Kabat numbering system: 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 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.

[0151] In some embodiments, any one or more amino acids of the anti-C1s antibodies described above are substituted with a histidine at the following positions according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96.

[0152] In some embodiments, the isolated anti-C1s antibody of the invention comprises one, two, three, four or five histidines substituted for amino acid residues at the following positions according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96.

[0153] In some embodiments, the isolated anti-C1s antibody of the invention comprises at least one histidine substituted residue at one or more of the following positions and CDR or FR amino acid positions according to the Kabat numbering system: Heavy chain: H51, H65, and H99; and Light chain: L92, L94, L95, and L96.

[0154] In some embodiments, the isolated anti-C1s antibody of the invention comprises at least one histidine substituted residue at the following positions according to the Kabat numbering system: 1) L92 and L94 2) L92 and L95 3) L94 and L95 4) L92, L94, and L95 5) H65 and L92 6) H65 and L94 7) H65 and L95 8) H65, L92, and L94 9) H65, L92, and L95 10) H65, L94, and L95 11) H65, L92, L94, and L95 12) H99 and L92 13) H99 and L94 14) H99 and L95 15) H99, L92, and L94 16) H99, L92, and L95 17) H99, L94, and L95 18) H99, L92, L94, and L95 19) H65 and H99 20) H65, H99, and L92 21) H65, H99, and L94 22) H65, H99, and L95 23) H65, H99, L92, and L94 24) H65, H99, L92, and L95 25) H65, H99, L94, and L95 26) H65, H99, L92, L94, and L95, or 27)H27, H99, and L95.

[0155] In any of the above-mentioned embodiments, the anti-C1s antibody is a humanized antibody. In one embodiment, the anti-C1s antibody comprises the HVR in any of the above-mentioned 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 the HVR in any of the above-mentioned embodiments and further comprises a VH or VL comprising a FR sequence.

[0156] In a further aspect, the present invention provides an antibody that binds to the same epitope as the anti-C1s antibody provided herein. For example, in a particular embodiment, the present invention provides an antibody that binds to the same epitope as an antibody selected from the group consisting of 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, IPN-M29, and IPN-M33 disclosed in WO2014 / 066744.

[0157] In some embodiments, an isolated anti-C1s antibody of the present invention competes for binding to C1s at neutral pH with an antibody selected from the group consisting of 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, IPN-M29, and IPN-M33 disclosed in WO2014 / 066744.

[0158] In one aspect, the present disclosure provides an isolated humanized monoclonal antibody having pH-dependent binding properties that specifically binds to an epitope within a region including domains IV and V of complement component Is (C1s). In some examples, the antibody inhibits binding of C1s to complement component 4 (C4). In some examples, the antibody does not inhibit the protease activity of C1s. In some examples, the epitope bound by the isolated humanized monoclonal antibody of the present disclosure is a conformational epitope.

[0159] In one aspect, the present disclosure provides an isolated antibody having pH-dependent binding that specifically binds to an epitope in complement C1s protein. In some embodiments, the isolated anti-C1s antibody of the present disclosure binds to activated C1s protein. In some embodiments, the isolated anti-C1s antibody of the present disclosure binds to the inactive form of C1s. In other examples, the isolated anti-C1s antibody of the present disclosure binds to both activated C1s protein and the inactive form of C1s.

[0160] In one embodiment, the present disclosure provides an isolated humanized monoclonal antibody having pH-dependent binding that specifically binds to an epitope within a region including 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 examples, 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 binding of C4 to C1s. The present disclosure also provides a method of treating a complement-mediated disease or disorder, 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 binding of C4 to C1s.

[0161] In one aspect, the present disclosure provides an isolated humanized monoclonal antibody having pH-dependent binding properties that specifically binds to an epitope comprising an aspartic acid at position 372 of human C1s antigen as shown in SEQ ID NO:3.

[0162] In a further aspect of the invention, the anti-C1s antibody according to any of the above embodiments is a monoclonal antibody, including a chimeric, humanized, or human antibody. In one embodiment, the anti-C1s antibody is an antibody fragment, such as, for example, an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, for example, a complete IgG1, IgG2, IgG3, or IgG4 antibody, or other antibody class or isotype as defined herein.

[0163] In further aspects, the anti-C1s antibody according to any of the above embodiments may incorporate, either alone or in combination, any of the features described in items 1-7 below.

[0164] 1. Antibody affinity In certain embodiments, the antibodies provided herein have a concentration 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 less, e.g. 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 The enzyme has a dissociation constant (Kd or KD) of 0.01 M.

[0165] In one embodiment, Kd is measured by radiolabeled antigen binding assay (RIA). In one embodiment, the RIA is performed using a Fab version of the antibody of interest and its antigen. For example, the solution binding affinity of the Fab for the antigen is determined by measuring the binding affinity of the Fab to the antigen at the lowest concentration ( 125I) Fab is equilibrated with labeled antigen, and then bound antigen is captured by a plate coated with anti-Fab antibody. (See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish the measurement conditions, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 μg / ml of 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 2-5 hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM of [ 125 I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., as in the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight, although the incubation can be continued for longer (e.g., about 65 hours) to ensure that equilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution is then removed and the plate is washed 8 times with 0.1% polysorbate 20 (TWEEN®-20) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT-20™, Packard) is added and the plate is counted for 10 minutes in a TOPCOUNT™ gamma counter (Packard). The concentration of each Fab that gives 20% or less of maximum binding is selected for use in the competitive binding assay.

[0166] In another embodiment, Kd is measured using a BIACORE® surface plasmon resonance assay. For example, an assay using a BIACORE®-2000 or BIACORE®-3000 (BIACORE®, Inc., Piscataway, NJ) is performed at 25° C. using a CM5 chip with approximately 10 response units (RU) of antigen immobilized. 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. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, before being injected at a flow rate of 5 μL / min to achieve approximately 10 response units (RU) of protein binding. After injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN®-20) surfactant (PBST) at 25° C. with a flow rate of approximately 25 μL / min. The binding rate (k on ) and dissociation rate (k off ) is calculated by simultaneously fitting the association and dissociation sensorgrams with a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2). The equilibrium dissociation constant (Kd) is k off / k on The on-rate is calculated as a ratio of 10 to 10 by the surface plasmon resonance assay described above. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). 6 M -1 s -1If the on-rate exceeds , the on-rate can be determined by using a fluorescence quenching technique to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, band pass 16 nm) 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 (e.g., a stopped-flow spectrophotometer (Aviv Instruments) or an 8000 series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) using a stirred cuvette).

[0167] In some embodiments, the binding affinity of each histidine substitution variant of the present 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). The 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 on the sensor surface by Protein A / G. The amount of antibody capture is typically targeted to 100-200 resonance units (RU). Serum-derived human C1s (CompTech) or prepared recombinant C1s can be injected at, for example, 50 or 200 nM, followed by dissociation. The sensor surface is regenerated every cycle, for example, with 10 mM glycine-HCl, pH 1.5. Binding affinity can be determined, for example, by processing data and fitting to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare).

[0168] A specific example of the steps of a Biacore assay of the present invention is as follows. The binding affinity of histidine-substituted variants 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) is immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes are prepared in 7(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4) or 5(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8). Each antibody is captured on the sensor surface by Protein A / G. The amount of antibody capture is typically targeted to 100-200 resonance units (RU). Serum-derived human C1s is injected at 12.5, 50 nM at pH 7.4, or at 50, 200 nM at pH 5.8, or at 200 and 800 nM at pH 5.8, followed by dissociation. After each cycle, the sensor surface is regenerated with 10 mM glycine-HCl, pH 1.5. The binding affinity is determined by processing and fitting the data to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare). An additional dissociation region at pH 5.8 is incorporated immediately after the dissociation region at pH 7.4. The dissociation rate in 5(+) buffer is determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software. Alternatively, the binding affinity of histidine-substituted variants 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) is immobilized on all flow cells of a CM4 sensor chip using an amine coupling kit (GE Healthcare). Antibodies and analytes are prepared in 7(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 7.4) or 5(+) buffer (20 mM ACES, 150 mM NaCl, 1.2 mM CaCl2, 0.05% Tween 20, 0.005% NaN3, pH 5.8). Each antibody is captured on the sensor surface by Protein A / G. The amount of antibody capture is typically targeted to 100-200 resonance units (RU). Serum-derived human C1s is injected at 50 nM and subsequently dissociated. After each cycle, the sensor surface is regenerated with 10 mM glycine-HCl, pH 1.5. The binding affinity is determined by processing and fitting the data to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0 (GE Healthcare). The dissociation region at pH 7.4 is immediately followed by an additional dissociation region at pH 5.8. The dissociation rate in 5(+) buffer is determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0169] In some embodiments, the analysis of the interaction between antibody and FcγR is carried out, for example, using BIACORE® T200 (GE Healthcare). 50 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride and 0.05 w / v% Tween-20 is used as the running buffer, and the measurement temperature is set to 25° C. A chip made by immobilizing Protein L (BioVision) on a Series S sensor chip CM4 (GE Healthcare) by amine coupling method is used. After capturing 500 or 1000 resonance units (RU) of the antibody of interest on the chip, FcγR diluted in running buffer is allowed to interact with it, and their binding amount to the antibody is measured. The antibody captured on the chip is then washed with 10 mM glycine HCl, pH 1.5, and the chip is regenerated and used repeatedly. The binding amount of FcγR is divided by the binding amount of each captured antibody to obtain the binding activity. In some embodiments, the analysis of the interaction between antibody and FcRn is carried out using BIACORE® T200 (GE Healthcare). A buffer solution containing 150 mM sodium chloride, 0.05 w / v% Tween-20, 50 mM phosphate buffer (pH 6.0) is used as the running buffer, and the measurement temperature is set to 25°C. A Series S sensor chip CM4 (GE Healthcare) with Protein L (BioVision) immobilized by amine coupling method is used. After capturing 400 resonance units (RU) of the antibody of interest on the chip through Protein L, it is allowed to interact with FcRn diluted with running buffer, and then its KD value is calculated. The chip with the captured antibody is then washed with 10 mM glycine HCl, pH 1.5, regenerated, and used repeatedly. Based on the steady-state analysis, the equilibrium dissociation constant (KD(M)) for the antibody to FcRn is calculated.

[0170] In some embodiments, an additional dissociation region at pH 5.8 is optionally incorporated immediately following the dissociation region at pH 7.4. This dissociation rate in 5(+) buffer can be determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software.

[0171] 2. Antibody fragment 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 specific antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.269-315 (1994); in addition, WO93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased half-lives in vivo.

[0172] Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); 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).

[0173] A single domain antibody is an antibody fragment that contains all or a portion of the heavy chain variable domain or all or a portion 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. Patent No. 6,248,516 B1).

[0174] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phages), as described herein.

[0175] 3. Chimeric and humanized antibodies In certain embodiments, the antibody provided herein is a chimeric antibody. 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 non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass has been changed from that of the parent antibody. Chimeric antibodies also include antigen-binding fragments thereof.

[0176] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while maintaining the specificity and affinity of the parent non-human antibody. Usually, a humanized antibody comprises one or more variable domains, in which the HVRs (e.g., CDRs (or portions thereof)) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), for example, to restore or improve the specificity or affinity of the antibody.

[0177] Humanized antibodies and methods for making them are reviewed in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008) and also see, e.g., 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 resurfacing); 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 a "guide selection" approach for FR shuffling).

[0178] Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using the "best-fit" method (see Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions (see 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 of FR libraries (Baca et al., J. Biol. Chem. 272:10678-10684 (2008)). (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0179] 4. Human antibodies In certain embodiments, the antibody provided herein is a human antibody. Human antibodies can be produced by various techniques known in the art. Human antibodies are reviewed in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0180] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been engineered to produce fully human antibodies or complete antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or a portion of a human immunoglobulin locus, which either replaces an endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosomes. 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, which describe XENOMOUSE™ technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. 2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from complete antibodies generated by such animals may be further modified, for example, by combining with different human constant regions.

[0181] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor, J. Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol. 147: 86 (1991).) Human antibodies generated via 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, for example, in U.S. Patent No. 7,189,826 (describing 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).

[0182] Human antibodies can also be generated by isolating Fv clone variable domain sequences selected from a human-derived phage display library. Such variable domain sequences can then be combined with the desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0183] 5. Library-derived antibodies Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are reviewed in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., 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 Molecular Biology 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).

[0184] In a particular phage display method, repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol. 12: 433-455 (1994). Phages typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies to a wide range of non-self and self antigens without immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can be made synthetically by cloning pre-rearranged V-gene segments from stem cells and using PCR primers that encode the hypervariable CDR3 regions and contain random sequences to achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol. 227: 381-388 (1992). Patent literature describing human antibody phage libraries includes, for example: U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0185] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0186] 6. Multispecific antibodies In certain embodiments, the antibodies provided herein are multispecific antibodies (e.g., bispecific antibodies). Multispecific antibodies are monoclonal antibodies that have binding specificities at 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 may bind to two different epitopes of C1s. Bispecific antibodies may be used to localize cytotoxic agents to cells expressing C1s. Bispecific antibodies may be prepared as full length antibodies or as antibody fragments.

[0187] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983), WO93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and the "knob-in-hole" technology (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can be made by manipulating electrostatic steering effects to create Fc heterodimeric molecules (WO2009 / 089004A1); cross-linking two or more antibodies or fragments (see U.S. Pat. No. 4,676,980 and Brennan et al., Science 229: 81 (1985)); using leucine zippers to generate antibodies with two specificities (see Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (Gruber et al., J. Immunol., 152:5368 (1994)). see above); and by preparing trispecific antibodies, for example, as described in Tutt et al., J. Immunol. 147: 60 (1991).

[0188] Engineered antibodies with three or more functional antigen binding sites are also included herein, including "octopus antibodies" (see, eg, US Patent Application Publication No. 2006 / 0025576 A1).

[0189] The antibody or fragment herein also includes a "dual-acting Fab" or "DAF" that contains one antigen-binding site that binds to C1s and another distinct antigen (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0190] 7. Antibody variants In certain embodiments, amino acid sequence variants of the antibodies provided herein are also contemplated. 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 may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., antigen binding).

[0191] a) Substitution, insertion, and deletion variants In certain embodiments, antibody variants with one or more amino acid substitutions are provided. Target sites for substitution mutations include HVR and FR. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". More substantial changes are provided in Table 1 under the heading of "exemplary substitutions" and are detailed below with reference to classes of amino acid side chains. Amino acid substitutions may be introduced into the antibody of interest, and the products may be screened for desired activity, such as, for example, retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0192] [Table 1]

[0193] Amino acids can be divided into groups according to common side chain properties: (1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile); (2) neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln); (3) Acidic: aspartic acid (Asp), glutamic acid (Glu); (4) Basic: histidine (His), lysine (Lys), arginine (Arg); (5) Residues that affect chain orientation: glycine (Gly), proline (Pro); (6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe). Non-conservative substitutions refer to the exchange of a member of one of these classes for one from another class.

[0194] One type of substitutional variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Usually, the resulting variant selected for further testing will have a modification (e.g., an improvement) in a particular biological property compared to the parent antibody (e.g., increased affinity, reduced immunogenicity) and / or will substantially retain a particular biological property of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, for example, using phage display-based affinity maturation techniques (e.g., those described herein). Briefly, one or more HVR residues are mutated and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0195] Modifications (e.g., substitutions) can be made in HVRs, for example, to improve antibody affinity. Such modifications can be made in "hot spots" of HVRs, i.e., residues encoded by codons where mutations occur frequently during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues that contact antigens, and the resulting variant VH or VL can be tested for binding affinity. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al., in 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. This library is then screened to identify any antibody variants with the desired affinity. Another method of introducing diversity involves HVR-directed approaches, where 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. In particular, CDR-H3 and CDR-L3 are often targeted.

[0196] In certain embodiments, substitution, insertion, or deletion can be made in one or more HVRs, as long as such modification does not substantially reduce the ability of antibody to bind to antigen.For example, conservative modifications (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in HVRs.Such modifications can be, for example, outside the antigen contact residues of HVRs.In certain embodiments of the above variant VH and VL sequences, each HVR is unmodified or contains only one, two, or three amino acid substitutions.

[0197] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is that described by Cunningham and Wells (1989), Science 244:1081-1085, called "alanine scanning mutagenesis." In this method, a residue or group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and it is determined whether the antibody-antigen interaction is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to this initial substitution. Alternatively or additionally, a crystal structure of the antigen-antibody complex can be analyzed to identify contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted as replacement candidates or excluded from replacement candidates. The variants can be screened to determine whether they contain the desired properties.

[0198] Amino acid sequence insertions include fusions ranging in length from one residue to polypeptides containing 100 or more residues at the amino and / or carboxyl termini, as well as single or multiple amino acid residue insertions within the sequence. An example of a terminal insertion includes an antibody with a methionyl residue at the N-terminus. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme (e.g., for ADEPT) or a polypeptide which increases the plasma half-life of the antibody.

[0199] b) Glycosylation variants In certain embodiments, the antibodies provided herein have been modified to increase or decrease the extent to which the antibody is glycosylated. Adding or deleting glycosylation sites to an antibody can be conveniently accomplished by modifying the amino acid sequence to create or remove one or more glycosylation sites.

[0200] If the antibody comprises an Fc region, the carbohydrate attached thereto may be modified. Natural antibodies produced by mammalian cells typically comprise branched, biantennary oligosaccharides, which are usually attached by N-linkage to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides include, for example, various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharides in the antibodies of the invention may be performed to generate antibody variants with specific improved properties.

[0201] In one embodiment, antibody variants are provided that have carbohydrate structures that lack fucose added (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1%-80%, 1%-65%, 5%-65% or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex, hybrid, and high mannose structures) added to Asn297, as measured by MALDI-TOF mass spectrometry, for example as described in WO2008 / 077546. Asn297 represents an asparagine residue located around position 297 of the Fc region (EU numbering of Fc region residues). However, due to slight sequence variability between multiple antibodies, Asn297 can also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include US2003 / 0157108; WO2000 / 61739; WO2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. 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, which lack protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 0157108 A1, Presta, L; and WO2004 / 056312 A1, Adams et al., especially Example 11) and knockout cell lines, such as alpha-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).

[0202] Further provided are antibody variants with bisected oligosaccharides, for example, bisected oligosaccharides added to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US2005 / 0123546 (Umana et al.). Also provided are antibody variants with at least one galactose residue in the oligosaccharide added to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997 / 30087 (Patel et al.); WO1998 / 58964 (Raju, S.); and WO1999 / 22764 (Raju, S.).

[0203] c) Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[0204] In certain embodiments, antibody variants with some, but not all, effector functions are also contemplated by the present invention, which make the antibody a desirable candidate for applications where its in vivo half-life is important, but certain effector functions (such as complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / lack of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks FcγR binding (and thus likely lacks ADCC activity) while retaining FcRn binding. NK cells, the primary cells mediating ADCC, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcR 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 to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 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); U.S. Pat. No. 5,821,337 (see, Bruggemann M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, e.g., ACT1™ non-radioactive cytotoxicity assays for flow cytometry (CellTechnology, Inc. Mountain View, Calif.); and CytoTox 96® non-radioactive cytotoxicity assays (Promega, Madison, Wis.)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind C1q and thus lacks CDC activity. See, e.g., C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. CDC measurements may also be performed to assess complement activation (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 et al., Blood 103:2738-2743 (2004)). In addition, FcRn binding and in vivo clearance / half-life determinations may also be performed using methods known in the art (see, e.g., Petkova SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0205] Antibodies with reduced effector function include those with one or more substitutions at Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant with substitutions of residues 265 and 297 to alanine (U.S. Patent No. 7,332,581).

[0206] Certain antibody variants with improved or decreased binding to FcRs have been described (see U.S. Pat. No. 6,737,056; WO2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0207] In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC (e.g., substitutions at positions 298, 333, and / or 334 (residues in EU numbering) of the Fc region).

[0208] In some embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or decreased) C1q 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).

[0209] Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) have been described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of 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., a substitution at Fc region residue 434 (U.S. Patent No. 7,371,826)).

[0210] For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351.

[0211] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies (e.g., "thioMAbs") in which one or more residues of an antibody are substituted with a cysteine ​​residue. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are placed at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to create immunoconjugates as further detailed herein. In certain embodiments, any one or more of the following residues may be substituted with a cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibodies may be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0212] e) antibody derivative In certain embodiments, the antibodies provided herein may be further modified to include additional non-protein moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies 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), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly 1,3 dioxolane, poly 1,3,6, trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody can vary, and if more than one polymer is attached, they can be the same molecule or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations such as, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in therapy under defined conditions, etc.

[0213] In another embodiment, a conjugate is provided between an antibody and a non-protein moiety that can be selectively heated by exposure to radiation. 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, including but not limited to wavelengths that heat the non-protein moiety to a temperature that is not harmful to normal cells but that kills cells in close proximity to the antibody-non-protein moiety.

[0214] B. Recombinant Methods and Constructs Antibodies can be produced using recombinant methods and constructs, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an anti-C1s antibody described herein. Such a nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., is transformed with) (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 eukaryotic (e.g., a Chinese hamster ovary (CHO) cell) or a lymphoid cell (e.g., a Y0, NS0, SP2 / 0 cell)). In one aspect, a method for producing an anti-C1s antibody is provided, comprising culturing a host cell containing a nucleic acid encoding the antibody as described above under conditions suitable for expression of the anti-C1s antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0215] For recombinant production of an anti-C1s antibody, nucleic acid encoding the antibody (such as those described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains).

[0216] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly if 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, which describes the expression of antibody fragments in E. coli.) After expression, the antibody may be isolated in a soluble fraction from the bacterial cell paste, and may be further purified.

[0217] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast, including fungal and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partial or fully human glycosylation patterns, are suitable cloning or expression hosts for antibody-encoding vectors. See Gerngross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006).

[0218] Host cells derived from multicellular organisms (invertebrate and vertebrate) are also suitable for the expression of glycosylated antibodies. Examples of invertebrate cells include plants and insect cells. Numerous baculovirus strains have been identified for conjugation with insect cells, particularly for transformation of Spodoptera frugiperda cells.

[0219] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing the PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0220] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that have been adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (293 or 293 cells, e.g., as described 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 hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary carcinoma (MMT 060562); TRI cells (e.g., as described in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)). Other useful mammalian host cell lines include DHFR cells, -Chinese hamster ovary (CHO) cells, including 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).

[0221] Antibodies with pH dependency can be obtained by using screening and / or mutagenesis methods, for example as described in WO 2009 / 125825. The screening method can include any process of identifying antibodies with pH-dependent binding properties from within a population of antibodies specific for a particular antigen. In certain embodiments, the screening method can include measuring one or more binding parameters (e.g., KD or kd) of individual antibodies in the initial antibody population at both acidic and neutral pH. The binding parameters of an antibody can be measured, for example, using surface plasmon resonance or any other analytical method that allows quantitative or qualitative evaluation of the binding properties of an antibody to a particular antigen. In certain embodiments, the screening method can include identifying antibodies that bind to an antigen with an acidic KD / neutral KD ratio of 2 or more. Alternatively, the screening method can include identifying antibodies that bind to an antigen with an acidic kd / neutral kd ratio of 2 or more.

[0222] In another embodiment, the mutagenesis method may include incorporating deletions, substitutions, or additions of amino acids in the heavy and / or light chains of the antibody to enhance the pH-dependent binding of the antibody to the antigen. In certain embodiments, the mutagenesis may be performed in one or more variable domains of the antibody, such as in one or more HVRs (e.g., CDRs). For example, the mutagenesis may include substituting an amino acid in one or more HVRs (e.g., CDRs) of the antibody with another amino acid. In certain embodiments, the mutagenesis may include substituting 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" means that the mutant antibody exhibits a greater acidic KD / neutral KD ratio or a greater acidic kd / neutral kd ratio than the original "parent" antibody (i.e., an antibody with low pH dependence) before mutagenesis. In certain embodiments, the mutant antibody has an acidic KD / neutral KD ratio of 2 or more. Alternatively, the mutant antibody has an acidic kd / neutral kd ratio of 2 or more.

[0223] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. The relevant antigen is conjugated to a protein that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, with a bifunctional substance or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R. 1 N=C=NR, where R and R 1 are different alkyl groups).

[0224] An animal (usually a non-human mammal) is immunized against the antigen, immunogenic conjugate, or derivative, for example, by 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 serum is assayed for antibody titer. The animal is boosted until the titer reaches a plateau. The animal is preferably boosted with the same antigen but conjugated to a different protein and / or via a different cross-linking reagent. Conjugates can also be prepared as protein fusions in recombinant cell culture. Agglutinating agents such as alum are also suitably used to enhance the immune response.

[0225] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for potential naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of separate antibodies.

[0226] For example, monoclonal antibodies can be produced using the hybridoma method first described in Kohler et al., Nature 256(5517):495-497 (1975). In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described hereinabove to induce 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.

[0227] The immunizing agent typically includes an antigen protein or a fusion variant thereof. In general, peripheral blood lymphocytes (PBLs) are used if cells of human origin are desired, and spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103).

[0228] Immortalized cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. The hybridoma cells thus produced are seeded and grown in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), substances that prevent the growth of HGPRT-deficient cells.

[0229] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred are mouse myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 cells (and their derivatives, such as X63-Ag8-653) available from the American Type Culture Collection, Manassas, Virginia, USA. For the production of human monoclonal antibodies, human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described (Kozbor et al., J Immunol. 133(6):3001-3005 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp. 51-63 (1987)).

[0230] The culture medium in which the hybridoma cells are growing is assayed for the production of monoclonal antibodies against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by 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 the Scatchard analysis of Munson, Anal Biochem. 107(1):220-239 (1980).

[0231] After the hybridoma cells that produce the antibody of the desired specificity, affinity, and / or activity are identified, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. Hybridoma cells can also be grown in vivo as tumors in mammals.

[0232] 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.

[0233] C. Assay The anti-C1s antibodies provided herein may be identified, screened, or characterized for physical / chemical properties and / or biological activity by a variety of assays known in the art.

[0234] 1. Binding and other assays In one aspect, the antibodies of the invention are tested for their antigen binding activity by known methods, such as ELISA, Western blot, and the like.

[0235] In another aspect, a competitive assay can be used to identify an antibody that competes with any of the anti-C1s antibodies described herein for binding to C1s. In certain embodiments, when such a competing antibody is present in excess, it blocks (e.g., reduces) the 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 a competing antibody binds to the same epitope (e.g., linear or conformational epitope) as that bound by any of the anti-C1s antibodies described herein. Detailed exemplary methods for mapping the epitope to which an antibody binds are provided in Morris (1996), "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In certain embodiments, such competitive assays may be performed at neutral pH conditions.

[0236] In an exemplary competitive assay, immobilized C1s is incubated in a solution containing a first labeled antibody (e.g., one of those described herein) that binds to C1s and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to C1s. The second antibody may be present in a hybridoma supernatant. As a control, immobilized C1s is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to C1s, excess unbound antibody is removed and the amount of label bound to immobilized C1s is measured. If the amount of label bound to immobilized C1s is substantially reduced in the test sample compared 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 ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0237] In another aspect, an antibody that binds to the same epitope as the anti-C1s antibody provided herein or competes with the anti-C1s antibody 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 can bind 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 ternary complex. See David & Greene, U.S. Pat. No. 4,376,110. The second antibody may itself be labeled with a detectable moiety (direct sandwich assay) or may 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 this example the detectable moiety is an enzyme. An antibody that binds to C1s simultaneously with the anti-C1s antibody provided herein can be determined to be an antibody that binds to a different epitope than the anti-C1s antibody. Therefore, an antibody that does not bind to C1s at the same time as the anti-C1s antibody provided in the present specification can be determined to be an antibody that binds to the same epitope as the anti-C1s antibody or competes with the anti-C1s antibody for binding to C1s.

[0238] 2. Activity assay In one aspect, an assay is provided for identifying anti-C1s antibodies with biological activity. Biological activity can include blocking activation of the classical pathway and blocking the generation of cleavage products C2a, C2b, C3a, C3b, C4a, C4b, C5a, and C5b resulting from activation of the classical pathway. Antibodies with such biological activity in vivo and / or in vitro are also provided.

[0239] In certain embodiments, the antibodies of the present invention are tested for such biological activity. In some embodiments, the antibodies of the present invention can be evaluated for their ability to inhibit complement-mediated hemolysis of chicken red blood cells (cRBCs) sensitized by antibodies against cRBC antigens. Using human serum as a source of complement proteins, the activity of the antibodies of the present invention can be determined by measuring the amount of released hemoglobin spectrophotometrically. In some embodiments, the antibodies of the present invention can be evaluated for their ability to inhibit cleavage of purified C4 (but not C2) via activated C1s. This activity of the antibodies is determined by measuring the amount of cleaved C4 or C2 by gel electrophoresis or Western blotting. Cleaved C4 or C2 can be detected by its smaller molecular weight compared to its native uncleaved form.

[0240] 3. Mouse PK study to evaluate antigen (C1s) removal In certain embodiments, promotion of clearance of an antigen (eg, human C1s (also referred to as hC1s)) by an antibody of the invention can be assessed in vivo (eg, in mice) as follows. Measurement of C1s concentrations in mouse plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry (LC / ESI-MS / MS) The concentration of hC1s (or anti-C1s antibodies) in mouse plasma can be measured by LC / ESI-MS / MS. A calibration standard is prepared by mixing and diluting a defined amount of hC1s (or anti-C1s antibodies) in mouse plasma. The calibration standard and plasma sample are mixed with urea, dithiothreitol and lysozyme (chicken egg white), for example in ammonium bicarbonate, and incubated. Iodoacetamide is then added and incubated in the dark. Trypsin in ammonium bicarbonate is then added and incubated. Finally, trifluoroacetic acid is added to inactivate residual trypsin. The sample is subjected to analysis by LC / ESI-MS / MS. Human C1s-specific peptides (e.g., LLEVPEGR) are monitored by selected reaction monitoring (SRM). The SRM transition can be [M+2H]2+ (m / z 456.8) to y6 ion (m / z 686.4) for human C1s. A calibration curve can be generated by weighted (1 / x2) linear regression with peak areas plotted against concentration, from which concentrations in mouse plasma are calculated. Evaluation of the pharmacokinetics of total hC1s following administration of anti-C1s antibody in mice The in vivo pharmacokinetics of hC1s, hC1q, or anti-C1s antibodies can be evaluated after administering antigen alone or together with anti-C1s antibodies to mice. A solution / mixture containing hC1s (etc.) is intravenously injected into mice. After administration of the antigen solution, the same individual is immediately administered with anti-C1s antibody solution in the same manner. The dose setting can be appropriately designed so that almost all of the hC1s is in bound form in the blood. Blood is collected over time, for example, 5, 30 minutes, 2, 7 hours, 3, 7, 14, 21, and 28 days after injection. The blood is immediately centrifuged to separate plasma samples. The plasma concentration of hC1s (etc.) is measured by LC / ESI-MS / MS at each sample collection time point. The PK parameters of hC1s (etc.) are estimated by non-compartmental analysis. For example, for anti-C1s antibodies, the hC1s CL (clearance) ratio is calculated. If this ratio is high, this means that hC1s removal can be more promoted.

[0241] 3-2. Monkey PK study to evaluate antigen clearance In certain embodiments, the promotion of antigen (eg, C1s and C1q) clearance by antibodies of the invention can be assessed in vivo (eg, in cynomolgus monkeys) as follows. Measurement of total cynomolgus monkey C1s and C1q concentrations in monkey plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry (LC / ESI-MS / MS) The total cynomolgus C1s concentration in plasma can be measured by LC / ESI-MS / MS. A calibration standard is prepared by mixing and diluting a defined amount of cynomolgus C1s in mouse plasma to obtain cynomolgus C1s concentrations of, for example, 0.954, 1.91, 3.82, 7.63, 15.3, 30.5, and 61.1 μg / mL. The cynomolgus plasma sample is diluted with mouse plasma. The calibration standard or the diluted plasma sample is mixed with, for example, urea, dithiothreitol, lysozyme (chicken egg white), and human C1s in ammonium bicarbonate in a ratio of 20:2:2.5:2, and incubated. Iodoacetamide is then added and incubated in the dark. Trypsin, for example, sequencing grade modified trypsin (Promega), is then added and incubated. Finally, trifluoroacetic acid is added to inactivate residual trypsin. The digested sample is subjected to analysis by LC / ESI-MS / MS. Cynomolgus C1q concentration in plasma is measured by LC / ESI-MS / MS. Calibration standards are prepared by mixing and diluting a defined amount of cynomolgus C1q in human plasma to give cynomolgus C1q concentrations of, for example, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, and 100 μg / mL. Cynomolgus plasma samples are diluted with human plasma. Calibration standards and diluted plasma samples are mixed with anti-C1q antibody and administered anti-C1s antibody in bovine serum albumin, respectively. Each sample is mixed with diluted Protein A magnetic beads (Protenova). After washing, the samples are mixed and incubated with, for example, urea, dithiothreitol, and lysozyme (chicken egg white) in ammonium bicarbonate in a ratio of 20:2:2.5. Iodoacetamide is then added and incubated in the dark. Sequencing grade modified trypsin (Promega) in ammonium bicarbonate is then added and incubated. Finally, trifluoroacetic acid is added to inactivate residual trypsin. The digested sample is subjected to analysis by LC / ESI-MS / MS.

[0242] [LC / ESI-MS / MS analysis] Peptides LLEVPEAR (SEQ ID NO: 32), LLEVPEGR (SEQ ID NO: 33), and YQSVFTVAR (SEQ ID NO: 34), which are specific for cynomolgus C1s, human C1s, and cynomolgus C1q, respectively, are monitored by selected reaction monitoring (SRM). The SRM transitions are [M+2H] 2+ (m / z 463.8~y6 1+ (m / z 700.3)), [M+2H] 2+ (m / z 456.8~y6 1+ (m / z 686.3)), and [M+2H] 2+ (m / z 535.8~y7 1+ (m / z 779.4). Each calibration curve is constructed by weighted (1 / x2) linear regression with peak areas plotted against concentration. Concentrations in cynomolgus monkey plasma are calculated from the calibration curve.

[0243] D. Immunoconjugates The present invention also provides immunoconjugates comprising the anti-C1s antibodies herein conjugated to one or more cytotoxic agents (e.g., a chemotherapeutic agent or drug, a growth inhibitory agent, a toxin (e.g., a protein toxin of bacterial, fungal, plant or animal origin, an enzymatically active toxin, or fragments thereof), or a radioactive isotope).

[0244] In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which an 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 No. 0,425,235 B1); auristatins, such as the monomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298); dolastatins; calicheamicin or a derivative thereof (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 see Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (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. Patent No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

[0245] 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, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0246] In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Examples include: 211 At, 131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Pb, and radioactive isotopes of Lu. When the radioconjugate is used for detection, the radioconjugate is a radioactive atom for scintigraphy (e.g., Tc-99m or 123 I), or spin labels (again, e.g., iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron) for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI).

[0247] Conjugates of antibodies and cytotoxic agents can be made using a variety of bifunctional protein linking 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 adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium 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 conjugation of radionuclides to antibodies. See WO94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020) can be used.

[0248] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, conjugates prepared using cross-linking reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0249] E. Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the anti-C1s antibodies provided herein is useful for detecting the presence of C1s in biological samples.The term "detection" as used herein includes quantitative or qualitative detection.In certain embodiments, the biological sample comprises cells or tissues, such as serum, whole blood, plasma, biopsy sample, tissue sample, cell suspension, saliva, sputum, oral fluid, cerebrospinal fluid, amniotic fluid, peritoneal fluid, breast milk, colostrum, mammary gland secretion, lymphatic fluid, urine, sweat, tears, gastric juice, synovial fluid, peritoneal fluid, ophthalmic lens fluid, or mucus.

[0250] In one embodiment, an anti-C1s antibody is provided for use in a diagnostic method or a detection method. In a further aspect, a method is provided for detecting the presence of C1s in a biological sample. In a particular embodiment, the method comprises contacting a biological sample with an anti-C1s antibody described herein under conditions that allow the binding of the anti-C1s antibody to C1s, and detecting whether a complex is formed between the anti-C1s antibody and C1s. Such a method can be an in vitro method or an in vivo method. In one embodiment, the anti-C1s antibody is used to select subjects that are suitable for treatment with the anti-C1s antibody, for example, when C1s is a biomarker for selecting patients.

[0251] Examples of disorders that may be diagnosed using the antibodies of the invention include age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, argyrophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uraemic syndrome, autoimmune diseases, Barraquer-Simons syndrome, Behcet's disease, British amyloid angiopathy, bullous pemphigoid, Buerger's disease, C1q nephropathy, cancer, fulminant antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Crohn's disease, cryoglobulinemic vasculitis, dementia pugilistica, dementia with Lewy bodies (DLB), diffuse neurofibrillary tangle disease with calcifications, discoid lupus erythematosus, Down's syndrome, focal segmental glomerular nephropathy, and glaucoma. Sclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia linked to chromosome 17 with parkinsonism, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker syndrome, Guillain-Barré syndrome, Hallervorden-Spatz syndrome, hemolytic uraemic syndrome, hereditary angioedema, hypophosphatasia, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infections (e.g. bacterial (e.g. meningococcal or streptococcal), viral (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 (MoCD) type A, membranoproliferative glomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis (MPGN) II (dense deposit disease), membranous nephritis, multi-infarct dementia, lupus (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-Guam 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 (E.These conditions include, but are not limited to, Stenotrophomonas erythropoietin (STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, neurofibrillary dementia, graft rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegener'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 refractory state.

[0252] In certain embodiments, labeled anti-C1s antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels) and indirectly detectable moieties (e.g., enzymes or ligands), for example, through enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, the following: radioisotopes 32 P, 14 C. 125 I, 3 H and 131I, fluorophores such as rare earth chelates or luciferases such as fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, monosaccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), linked to enzymes that oxidize dye precursors using hydrogen peroxide (e.g., HRP, lactoperoxidase, or microperoxidase), heterocyclic oxidases such as uricase and xanthine oxidase, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0253] F. Pharmaceutical Formulations Pharmaceutical formulations of the antigen-binding molecules described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing the molecule having the desired purity with any one or more pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, the following: buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.; small (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, sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmacologic carriers herein further include interstitial drug dispersing agents, such as soluble neutral activated hyaluronidase glycoproteins (sHASEGPs) (e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.)). Certain exemplary sHASEGPs and methods of their use (including rHuPH20) are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as a chondroitinase.

[0254] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0255] The formulations herein may contain more than one active ingredient if necessary for the specific indications to be treated. Those with complementary activities that do not adversely affect each other are preferred. For example, it may be desirable to provide further formulations for use in combination therapy. Such active ingredients are present in suitable combinations in amounts that are effective for the intended purpose.

[0256] The active ingredient may be incorporated into microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by droplet formation (coacervation) techniques or by interfacial polymerization, into colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or into macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0257] 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, eg, films, or microcapsules.

[0258] Preparations to be used for in vivo administration are typically sterile. Sterility is readily accomplished, for example, by filtration through sterile filtration membranes.

[0259] G. Therapeutic Methods and Compositions Any of the antigen binding molecules provided herein may be used in therapeutic methods. In one aspect, an antigen-binding molecule is provided for use as a medicament. In a further aspect, an antigen-binding molecule is provided for use in the treatment of a complement-mediated disease or disorder. In a particular embodiment, an antigen-binding molecule is provided for use in a method of treatment. In a particular embodiment, the present invention provides an antigen-binding molecule for use in a method of treating an individual with a complement-mediated disease or disorder, the method comprising administering to the individual an effective amount of the antigen-binding molecule. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent. The "individual" according to any of the above embodiments is preferably a human.

[0260] In a further embodiment, the present invention provides an antigen binding molecule for use in treating a complement-mediated disease or disorder. In a further embodiment, the antigen binding molecule of the present invention can be for use in enhancing the clearance of C1s from plasma. In a further embodiment, the antigen binding molecule of the present invention can be for use in enhancing the clearance of a complex of C1q, C1r and C1s from plasma. In a further embodiment, the antigen binding molecule of the present invention can be for use in inhibiting the cleavage of complement component C4, where the molecule does not inhibit the cleavage of complement component C2. In some examples, the molecule inhibits a component of the classical complement pathway, and in some examples, the component of the classical complement pathway is C1s. In a particular embodiment, the present invention provides an antigen binding molecule for use in a method of treating a complement-mediated disease or disorder. In a particular embodiment, the present invention provides an antigen binding molecule for use in a method of enhancing the clearance of C1s from plasma. In a particular embodiment, the present invention provides an antigen binding molecule for use in a method of enhancing the clearance of a complex of C1q, C1r and C1s from plasma. In certain embodiments, the present invention provides an antigen binding molecule for use in a method for inhibiting cleavage of complement component C4, which does not inhibit cleavage of complement component C2. In certain embodiments, the present invention provides an antigen binding molecule for use in a method for inhibiting a component of the classical complement pathway, and in some examples, the component of the classical complement pathway is C1s. In any of the above embodiments, the "individual" is preferably a human.

[0261] In one aspect, the present disclosure provides a method of modulating 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 of modulating complement activation in an individual with a complement-mediated disease or disorder, comprising administering to the individual an antigen binding molecule of the present disclosure or a pharmaceutical composition of the present disclosure, wherein the pharmaceutical composition comprises an antigen binding molecule of the present disclosure. In some embodiments, such a 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 or subcutaneous administration. In some embodiments, administration is intrathecal administration.

[0262] A complement-mediated disease or disorder is a disorder characterized by abnormal amounts of complement C1s or abnormal levels of complement C1s proteolytic activity in the cells, tissues, or body fluids of an individual.

[0263] In some instances, a complement-mediated disease or disorder is characterized by the presence of an increased (higher than normal) amount of C1s or an elevated level of complement C1s activity in cells, tissues, or body fluids. For example, in some instances, a complement-mediated disease or disorder is characterized by the presence of an increased amount of C1s and / or elevated activity of C1s in brain tissue and / or cerebrospinal fluid. A "higher than normal" amount of C1s in a cell, tissue, or body fluid indicates that the amount of C1s in that cell, tissue, or body fluid is higher than the normal control level, e.g., higher than the normal control level of an individual or population of individuals of the same age group. A "higher than normal" level of C1s activity in a cell, tissue, or body fluid indicates that the proteolytic cleavage caused by C1s in that cell, tissue, or body fluid is higher than the normal control level, e.g., higher than the normal control level of an individual or population of individuals of the same age group. In some instances, an individual with a complement-mediated disease or disorder exhibits one or more additional symptoms of such disease or disorder.

[0264] In other examples, a complement-mediated disease or disorder is characterized by the presence of a lower amount of C1s or a lower level of complement C1s activity in cells, tissues, or body fluids than normal. For example, in some examples, a complement-mediated disease or disorder is characterized by the presence of a lower amount of C1s and / or a lower activity of C1s in brain tissue and / or cerebrospinal fluid. A "lower than normal" amount of C1s in a cell, tissue, or body fluid indicates that the amount of C1s in that cell, tissue, or body fluid is lower than a normal control level, e.g., lower than a normal control level for an individual or population of individuals of the same age group. A "lower than normal" level of C1s activity in a cell, tissue, or body fluid indicates that the proteolytic cleavage caused by C1s in that cell, tissue, or body fluid is lower than a normal control level, e.g., lower than a normal control level for an individual or population of individuals of the same age group. In some examples, an individual with a complement-mediated disease or disorder exhibits one or more additional symptoms of such disease or disorder.

[0265] A complement-mediated disease or disorder is a disease or disorder in which the amount or activity of complement C1s is sufficient to cause a disease or disorder in an individual. In some embodiments, the complement-mediated disease or disorder is selected from the group consisting of an autoimmune disease, a cancer, a blood disease, an infectious disease, an inflammatory disease, an ischemia-reperfusion injury, a neurodegenerative disease, a neurodegenerative disorder, an eye disease, a kidney disease, a transplant rejection, a vascular disease, and a vasculitic disease. In some embodiments, the complement-mediated disease or disorder is an autoimmune disease. In some embodiments, the complement-mediated disease or disorder is a cancer. In some embodiments, the complement-mediated disease or disorder is an infectious disease. In some embodiments, the complement-mediated disease or disorder is an inflammatory disease. In some embodiments, the complement-mediated disease or disorder is a blood disease. In some embodiments, the complement-mediated disease or disorder is an ischemia-reperfusion disease. In some embodiments, the complement-mediated disease or disorder is an eye disease. In some embodiments, the complement-mediated disease or disorder is a kidney disease. In some embodiments, the complement mediated disease or disorder is graft rejection. In some embodiments, the complement mediated disease or disorder is antibody mediated graft rejection. In some embodiments, the complement mediated disease or disorder is a vascular disease. In some embodiments, the complement mediated disease or disorder is a vasculitic disease. In some embodiments, the complement mediated disease or disorder is a neurodegenerative disease or disorder. In some embodiments, the complement mediated disease is a neurodegenerative disease. In some embodiments, the complement mediated disorder is a neurodegenerative disorder. In some embodiments, the complement mediated disease or disorder is a tauopathy.

[0266] Examples of complement-mediated diseases or disorders include age-related macular degeneration, Alzheimer's disease, amyotrophic lateral sclerosis, anaphylaxis, argyrophilic grain dementia, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, atypical hemolytic uremic syndrome, autoimmune diseases, Barraquer-Simons syndrome, and the like. syndrome), Behçet's disease, British amyloid angiopathy, bullous pemphigoid, Buerger's disease, C1q nephropathy, cancer, fulminant antiphospholipid syndrome, cerebral amyloid angiopathy, cold agglutinin disease, corticobasal degeneration, Creutzfeldt-Jakob disease, Crohn's disease, cryoglobulinemic vasculitis, dementia pugilistica, dementia with Lewy bodies (DLB), diffuse neurofibrillary tangle disease with calcification, discoid lupus erythematosus, Down's syndrome, focal segmental glomerular Sclerosis, formal thought disorder, frontotemporal dementia (FTD), frontotemporal dementia linked to chromosome 17 with parkinsonism, frontotemporal lobar degeneration, Gerstmann-Straussler-Scheinker syndrome, Guillain-Barré syndrome, Hallervorden-Spatz syndrome, hemolytic uraemic syndrome, hereditary angioedema, hypophosphatasia, idiopathic pneumonia syndrome, immune complex disease, inclusion body myositis, infections (e.g. bacterial (e.g. meningococcal or streptococcal), viral (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 (MoCD) type A, membranoproliferative glomerulonephritis (MPGN) I, membranoproliferative glomerulonephritis (MPGN) II (dense deposit disease), membranous nephritis, multi-infarct dementia, lupus (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-Guam 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 (E.These conditions include, but are not limited to, Stenotrophomonas erythropoietin (STEC)-HuS, spinal muscular atrophy, stroke, subacute sclerosing panencephalitis, neurofibrillary dementia, graft rejection, vasculitis (e.g., ANCA-associated vasculitis), Wegener'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 refractory state.

[0267] Alzheimer's disease and certain forms of frontotemporal dementia (Pick's disease, sporadic frontotemporal dementia, and frontotemporal dementia with parkinsonism linked to chromosome 17) are the most common forms of tauropathy. Thus, the present invention relates to any of the above methods, in which the tauropathy is Alzheimer's disease, Pick's disease, sporadic frontotemporal dementia, and frontotemporal dementia with parkinsonism linked to chromosome 17. Other tauropathy includes, but is not limited to, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and subacute sclerosing panencephalitis.

[0268] Neurodegenerative tauropathies include Alzheimer's disease, amyotrophic lateral sclerosis / Parkinsonism complex, argyrophilic grain dementia, British amyloid angiopathy, cerebral amyloid angiopathy, corticobasal degeneration, Creutzfeldt-Jakob disease, dementia pugilistica, diffuse neurofibrillary tangle disease with calcifications, Down's syndrome, frontotemporal dementia, frontotemporal dementia linked to chromosome 17 with parkinsonism, frontotemporal lobar degeneration, and Gerstmann-Sträussler-Scheinker syndrome. These include: Hallervorden-Spatz syndrome, inclusion body myositis, multiple system atrophy, myotonic dystrophy, Niemann-Pick disease type C, non-Guam motor neuron disease with neurofibrillary tangles, Pick's disease, postencephalitic Parkinson's disease, prion protein cerebral amyloid angiopathy, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, neurofibrillary dementia, multi-infarct dementia, ischemic stroke, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), and stroke.

[0269] The present disclosure also provides methods of treating synucleinopathies, such as Parkinson's disease (PD); dementia with Lewy bodies (DLB); multiple system atrophy (MSA), etc. For example, PD with dementia (PDD) can be treated using the methods of the present disclosure.

[0270] In some embodiments, the complement mediated disease or disorder comprises Alzheimer's disease. In some embodiments, the complement mediated disease or disorder comprises Parkinson's disease. In some embodiments, the complement mediated disease or disorder comprises transplant rejection. In some embodiments, the complement mediated disease or disorder is antibody mediated transplant rejection.

[0271] 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 disorder 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 disorder in an individual. Examples of symptoms include, but are not limited to, symptoms associated with autoimmune disease, cancer, blood disease, infectious disease, inflammatory disease, ischemia-reperfusion disease, neurodegenerative disease, neurodegenerative disorder, kidney disease, transplant rejection, eye disease, vascular disease, or vasculitis disease. The symptoms can be neurological symptoms, such as cognitive impairment, memory impairment, loss of motor function, and the like. The symptoms can also be the activity of C1s protein in the cells, tissues, or body fluids of the individual. The symptoms can also be the degree of complement activation in the cells, tissues, or body fluids of the individual.

[0272] In some embodiments, administration of the antigen-binding molecule of the present disclosure to an individual modulates complement activation in the cells, tissues, or body fluids of the individual. In some embodiments, administration of the antigen-binding molecule of the present disclosure to an individual inhibits complement activation in the cells, tissues, or body fluids of the individual. For example, in some embodiments, the antigen-binding molecule of the present disclosure, when administered in one or more doses as a monotherapy or in a combination therapy to an individual with a complement-mediated disease or disorder, inhibits 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 the complement activation in the individual before treatment with the antigen-binding molecule.

[0273] In some embodiments, the antigen binding molecules of the present disclosure reduce C3 deposition on red blood cells, e.g., in some embodiments, the antigen binding molecules of the present disclosure reduce deposition of C3b, iC3b, etc. on RBCs. In some embodiments, the antigen binding molecules of the present disclosure inhibit complement-mediated red blood cell lysis.

[0274] In some embodiments, the antigen binding molecules of the present disclosure reduce the deposition of C3 on platelets. For example, in some embodiments, the antigen binding molecules of the present disclosure reduce the deposition of C3b, iC3b, etc. on platelets.

[0275] In some embodiments, administration of an antigen binding molecule of the present disclosure results in an outcome selected from the group consisting of: (a) reduced complement activation, (b) improved cognitive function, (c) reduced neuronal loss, (d) reduced phosphorylated Tau levels in neurons, (e) reduced glial cell 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, (l) reduced neutrophil infiltration, (m) reduced red blood cell lysis, (n) reduced red blood cell activation. (o) reduced leukocyte phagocytosis, (p) reduced platelet phagocytosis, (q) improved graft survival, (r) reduced macrophage mediated phagocytosis, (s) improved vision, (t) improved motor control, (u) improved thrombus formation, (v) improved coagulation, (w) improved renal function, (x) reduced antibody mediated complement activation, (y) reduced autoantibody mediated complement activation, (z) improved anemia, (aa) reduced demyelination, (ab) reduced eosinophilia, (ac) reduced C3 deposition on red blood cells (e.g. reduced deposition of C3b, iC3b, etc. on RBCs), and and (ad) reduced C3 deposition on platelets (e.g. reduced C3b, iC3b, etc. deposition on platelets), and (ae) reduced anaphylatoxin production, (af) reduced autoantibody mediated blister formation, (ag) reduced autoantibody induced pruritus, (ah) reduced autoantibody induced lupus erythematosus, (ai) reduced autoantibody mediated skin erosions, (aj) reduced red blood cell destruction due to transfusion reactions, (ak) reduced red blood cell lysis due to alloantibody, (al) reduced hemolysis due to transfusion reactions, (am) reduced alloantibody mediated (an) reduced platelet lysis due to transfusion reactions, (ao) reduced mast cell activation, (ap) reduced mast cell histamine release, (aq) reduced vascular permeability, (ar) reduced edema, (as) reduced complement deposition on graft endothelium, (at) reduced anaphylatoxin production in graft endothelium, (au) reduced separation of the dermo-epidermal junction, (av) reduced anaphylatoxin production at the dermo-epidermal junction, (aw) reduced alloantibody-mediated complement activation in graft endothelium, (ax) reduced antibody-mediated loss of neuromuscular junctions.(ay) reduced complement activation at the neuromuscular junction, (az) reduced anaphylatoxin production at the neuromuscular junction, (ba) reduced complement deposition at the neuromuscular junction, (bb) reduced paralysis, (be) reduced numbness, (bd) improved bladder control, (be) improved bowel management, (bf) reduced autoantibody associated mortality, and (bg) reduced autoantibody associated morbidity.

[0276] In some embodiments, the antigen binding molecules of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, promote the following outcomes: (a) complement activation; (b) cognitive decline; (c) neuronal loss; (d) phosphorylated 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; (p) erythrocyte lysis; (n) erythrocyte phagocytosis; (o) platelet phagocytosis; (p) erythrocyte lysis ...lysis; (p) erythrocyte phagocytosis; (p) erythrocyte lysis; (p) erythrocyte lysis; (p) erythrocyte lysis; (p) erythrocyte lysis; (p) erythrocyte lysis; (p) erythrocyte phagocytosis; (p) erythrocyte (q) graft rejection; (r) macrophage-mediated phagocytosis; (s) vision loss; (t) antibody-mediated complement activation; (u) autoantibody-mediated complement activation; (v) demyelination; (w) eosinophilia 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 greater than 90%, compared to the level or degree of the outcome in the individual prior to treatment with the antigen-binding molecule.

[0277] In some embodiments, the antigen binding molecules of the present disclosure, when administered in one or more doses as monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, are effective in improving one or more of the following outcomes: a) cognitive function; b) graft survival; c) vision; d) motor control; e) clot formation; f) coagulation; g) renal function, and h) hematocrit (red blood cell count) 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 or degree of the outcome in the individual prior to treatment with the antigen binding molecule.

[0278] In some embodiments, administration of the antigen binding molecule of the present disclosure to an individual reduces complement activation in the individual. For example, in some embodiments, the antigen binding molecule of the present disclosure, when administered in one or more doses as a monotherapy or in a combination therapy to an individual with a complement-mediated disease or disorder, 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 the complement activation in the individual before treatment with the antigen binding molecule.

[0279] In some embodiments, administration of the antigen binding molecule of the present disclosure improves cognitive function in an individual. For example, in some embodiments, the antigen binding molecule of the present disclosure, when administered in one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, improves cognitive function 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 cognitive function in the individual before treatment with the antigen binding molecule.

[0280] In some embodiments, administration of the antigen binding molecule of the present disclosure reduces the rate of cognitive decline in an individual. For example, in some embodiments, the antigen binding molecule of the present disclosure, when administered in one or more doses as a monotherapy or in a combination therapy to an individual with a complement-mediated disease or disorder, reduces the rate of cognitive decline 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 rate of cognitive decline in the individual before treatment with the antigen binding molecule.

[0281] In some embodiments, administration of the antigen binding molecule of the present disclosure to an individual reduces neuronal loss in the individual.For example, in some embodiments, the antigen binding molecule of the present disclosure, when administered in one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, reduces neuronal loss 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 neuronal loss in the individual before treatment with the antigen binding molecule.

[0282] In some embodiments, administration of the antigen binding molecule of the present disclosure to an individual reduces the phosphorylated Tau level in the individual.For example, in some embodiments, the antigen binding molecule of the present disclosure, when administered in one or more doses to an individual with a complement-mediated disease or disorder as a monotherapy or in a combination therapy, reduces the phosphorylated 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 phosphorylated Tau level in the individual before treatment with the antigen binding molecule.

[0283] In some embodiments, administration of the antigen binding molecule of the present disclosure to an individual reduces glial cell activation in the individual.For example, in some embodiments, the antigen binding molecule of the present disclosure, when administered in one or more doses as a monotherapy or in combination therapy to an individual with a complement-mediated disease or disorder, reduces glial 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 the glial cell activation in the individual before treatment with the antigen binding molecule.In some embodiments, the glial cell is an astrocyte or a microglia.

[0284] In some embodiments, administration of the antigen binding molecule of the present disclosure to an individual reduces lymphocytic infiltration in the individual. For example, in some embodiments, the antigen binding molecule of the present disclosure, when administered in one or more doses as a monotherapy or in a combination therapy to an individual with a complement-mediated disease or disorder, reduces lymphocytic 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%, compared to the lymphocytic infiltration in the individual before treatment with the antigen binding molecule.

[0285] In some embodiments, administration of 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 in one or more doses as a monotherapy or in a combination therapy to an individual with a complement-mediated disease or disorder, 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%, compared to macrophage infiltration in the individual before treatment with the antigen binding molecule.

[0286] In some embodiments, administration of the antigen-binding molecule of the present disclosure to an individual reduces antibody deposition in the individual. For example, in some embodiments, the antigen-binding molecule of the present disclosure, when administered in one or more doses as a monotherapy or in a combination therapy to an individual with a complement-mediated disease or disorder, 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.

[0287] In some embodiments, administration of an antigen-binding molecule of the present disclosure to an individual reduces anaphylatoxin (e.g., C3a, C4a, C5a) production in the individual. For example, in some embodiments, an antigen-binding molecule of the present disclosure, when administered in one or more doses as a monotherapy or in a combination therapy to an individual with a complement-mediated disease or disorder, reduces anaphylatoxin production 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 anaphylatoxin production level in the individual before treatment with the antigen-binding molecule.

[0288] 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 pharma- ceutically acceptable excipient for treating an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides the use of an anti-C1s antibody of the present disclosure for treating an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides the use of a pharmaceutical composition comprising an antigen-binding molecule of the present disclosure and a pharma- ceutically acceptable excipient for treating an individual having a complement-mediated disease or disorder.

[0289] In some embodiments, the present disclosure provides the use of an antigen-binding molecule of the present disclosure in the manufacture of a medicament for treating an individual having a complement-mediated disease or disorder.

[0290] 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 pharma- ceutical acceptable excipient for inhibiting complement activation. 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 pharma- ceutical acceptable excipient for inhibiting complement activation in an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides the use of an antigen binding molecule of the present disclosure for inhibiting complement activation in an individual having a complement-mediated disease or disorder. In some embodiments, the present disclosure provides the use of a pharmaceutical composition comprising an antigen binding molecule of the present disclosure and a pharma- ceutical acceptable excipient for inhibiting complement activation in an individual having a complement-mediated disease or disorder.

[0291] In some embodiments, the present disclosure provides the use of an antigen binding molecule of the present disclosure in the manufacture of a medicament for modulating complement activation. In some embodiments, the medicament inhibits complement activation. In some embodiments, the medicament inhibits complement activation in an individual with a complement-mediated disease or disorder.

[0292] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antigen-binding molecule of the present disclosure or an antigen-binding molecule of the present disclosure and a pharmaceutically acceptable excipient for use in drug therapy. In some embodiments, the present disclosure provides an antigen-binding molecule of the present disclosure for use in drug therapy. 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 use in drug therapy.

[0293] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antigen-binding molecule of the present disclosure or an antigen-binding molecule of the present disclosure and a pharma- ceutically acceptable excipient for use in treating an individual with a complement-mediated disease or disorder. In some embodiments, the present disclosure provides an antigen-binding molecule of the present disclosure for use in treating an individual with a complement-mediated disease or disorder. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antigen-binding molecule of the present disclosure and a pharma- ceutically acceptable excipient for use in treating an individual with a complement-mediated disease or disorder.

[0294] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antigen binding molecule of the present disclosure or an antigen binding molecule of the present disclosure and a pharma- ceutical acceptable excipient for use in regulating complement activation. In some embodiments, the present disclosure provides an antigen binding molecule of the present disclosure for use in regulating complement activation. In some embodiments, the present disclosure provides a pharmaceutical composition comprising an antigen binding molecule of the present disclosure and a pharma- ceutical acceptable excipient for use in regulating complement activation. In some embodiments, the antigen binding molecule inhibits complement activation.

[0295] In a further aspect, the present invention provides the use of an antigen-binding molecule in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of a complement-mediated disease or disorder. In a further embodiment, the medicament is for use in a method of treating a complement-mediated disease or disorder, comprising administering an effective amount of the medicament to an individual having a complement-mediated disease or disorder. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional therapeutic agent (such as those described below). In a further embodiment, the medicament is for use in enhancing the clearance (or removal) of C1s from plasma. In a further embodiment, the medicament is for use in enhancing the clearance (or removal) of the complex of C1q, C1r, and C1s from plasma. In a further embodiment, the medicament is for use in inhibiting the cleavage of complement component C4, wherein the molecule does not inhibit the cleavage of complement component C2. In a further embodiment, the medicament is for use in inhibiting a component of the classical complement pathway, and in some instances, the component of the classical complement pathway is C1s.

[0296] In a further aspect, the medicament is for use in a method of treating an individual having a complement-mediated disease or disorder comprising administering to the individual an effective amount of the medicament. In any of the above aspects, the "individual" may be a human.

[0297] In a further aspect, the present invention provides a method for treating a complement-mediated disease or disorder. In one embodiment, the method comprises administering an effective amount of an antigen-binding molecule to an individual having such a complement-mediated disease or disorder. In one such embodiment, the method further comprises administering an effective amount of at least one additional therapeutic agent (described below) to the individual. In any of the above-mentioned embodiments, the "individual" can be a human.

[0298] In a further aspect, the present invention provides a method for enhancing the clearance (or removal) of C1s from plasma in an individual. In a further aspect, the present invention provides a method for enhancing the clearance (or removal) of a complex of C1q, C1r, and C1s from plasma in an individual. In some embodiments, the antigen binding molecule of the present invention provides a method in which the molecule inhibits cleavage of complement component C4 in an individual without inhibiting cleavage of complement component C2. In some examples, the present invention provides a method for inhibiting a component of the classical complement pathway in an individual, and in some examples, the component of the classical complement pathway is C1s. In one embodiment, the "individual" is a human.

[0299] In a further aspect, the present invention provides a pharmaceutical formulation comprising any of the antigen-binding molecules provided herein, for example for use in any of the above-mentioned therapeutic methods.In one embodiment, the pharmaceutical formulation comprises any of the antigen-binding molecules provided herein and a pharma- ceutically acceptable carrier.In another embodiment, the pharmaceutical formulation comprises any of the antigen-binding molecules provided herein and at least one additional therapeutic agent (e.g., as described below).

[0300] The antibodies of the invention can be used in therapy either alone or in combination with other agents, for example, the antibodies of the invention can be co-administered with at least one additional therapeutic agent.

[0301] Combination therapy as described above includes combined administration (two or more therapeutic agents are contained in the same or separate formulations) and separate administration, in which the administration of the molecule of the present invention can precede, be simultaneous with, and / or be subsequent to the administration of the additional therapeutic agent. In one embodiment, the administration of the antigen-binding molecule and the administration of the additional therapeutic agent are performed 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 molecule of the present invention can be used in combination with radiation therapy.

[0302] The antigen-binding molecules of the present invention (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, pulmonary, and nasal administration, and, if desired for localized treatment, intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. Various administration schedules are contemplated herein, including, but not limited to, single administration or repeated administration over various time periods, bolus administration, and pulse infusion.

[0303] The antigen-binding molecules of the present invention are formulated, administered, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration, and other factors known to medical practitioners. The molecule is optionally, but not necessarily, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of molecule present in the formulation, the type of disorder or treatment, and other factors discussed above. These are typically used in the same dosages and routes of administration as described herein, or about 1 to 99% of the dosages described herein, or any dosage and any route deemed empirically / clinically appropriate.

[0304] For the prevention or treatment of disease, the appropriate dose of the antigen-binding molecule of the present invention (when used alone or with one or more other additional therapeutic agents) will depend on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the molecule is administered for prophylactic or therapeutic purposes, medical history, the patient's clinical history and response to the molecule, and the discretion of the attending physician. The molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, for example, about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) of the molecule may be the initial candidate dose for administration to the patient, whether by one or more separate administrations or by continuous infusion. A typical daily dose may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. In the case of repeated administration over several days or longer, depending on the circumstances, treatment is usually maintained until a desired suppression of disease symptoms occurs. One exemplary dose of the molecule is in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, 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 intermittently, for example, every week or every three weeks (e.g., such that the patient receives about 2 to about 20, or, for example, about 6 doses of the molecule). A high initial loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0305] It will be appreciated that any of the above-described formulations or therapeutic methods may be practiced using the immunoconjugates of the invention in place of, or in addition to, antigen-binding molecules.

[0306] H. Other Methods In one aspect, the present invention provides a method for incorporating C1s or C1q into cells. In this aspect, C1s and cells are mixed in an isotonic solution. The cells express Fcγ receptors. This method includes adding any one of the above-mentioned antigen-binding molecules to the isotonic solution.

[0307] In one aspect, the present invention provides a method for reducing plasma C1s and / or C1q concentrations in an individual, comprising administering to the individual any one of the antigen-binding molecules described above.

[0308] I.Product In another aspect of the present invention, an article of manufacture is provided that includes materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders. The article of manufacture includes a container and a label on the container or a package insert associated with the container. Preferred containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The containers may be formed from a variety of materials, such as glass or plastic. The container may hold the composition alone or in combination with another composition effective for the treatment, prevention, and / or diagnosis of a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle). At least one active ingredient in the composition is an antigen-binding molecule of the present invention. The label or package insert indicates that the composition is used to treat the condition of choice. The article of manufacture may further comprise: (a) a first container with a composition comprising an antigen-binding molecule of the present invention contained therein; and (b) a second container with a composition comprising an additional cytotoxic or otherwise therapeutic agent contained therein. The article of manufacture in this aspect of the invention may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively or additionally, the article of manufacture may further comprise a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other equipment desirable from a commercial or user standpoint, such as other buffers, diluents, filters, needles, and syringes.

[0309] It will be appreciated that any of the above-mentioned articles of manufacture may contain an immunoconjugate of the invention instead of, or in addition to, an antigen-binding molecule. EXAMPLES

[0310] IV. Working Examples The following are examples of the methods and compositions of the present invention. In light of the general description above, it will be understood that various other embodiments may be practiced.

[0311] The foregoing invention has been described in detail by way of illustration and example for purposes of facilitating a clear understanding, but the descriptions and illustrations herein should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated herein by reference in their entireties.

[0312] Example 1 Preparation of recombinant C1r2s2 1.1. Expression and purification of cynomolgus C1r2s2 His / Flag tetramer The sequences used for expression and purification are cynomolgus C1s with a GGGGS linker and a Flag tag at the C-terminus (SEQ ID NO: 1) and cynomolgus C1r with a GGGGS linker and an 8x histidine tag at the C-terminus. The cynomolgus C1r sequence has the R463Q S654A mutation (SEQ ID NO: 2). For expression of recombinant cynomolgus C1r2s2 His / Flag tetramer, cynomolgus C1s-FLAG and cynomolgus C1r-His were transiently co-expressed using the FreeStyle® 293-F cell line (Thermo Fisher, Carlsbad, CA, USA). The culture supernatant expressing recombinant cynomolgus C1r2s2 His / FLAG tetramer was applied to anti-FLAGM2 affinity resin (Sigma) and eluted with FLAG peptide (Sigma). Fractions containing recombinant cynomolgus C1r2s2 His / FLAG tetramers were applied to an IMAC column (GE healthcare) and eluted with an imidazole gradient. The eluted fractions containing recombinant C1r2s2 His / FLAG tetramers were collected, concentrated, and then applied to a Superdex 200 gel filtration column (GE healthcare) equilibrated with 1x TBS, 2 mM CaCl2 buffer. Fractions containing recombinant cynomolgus C1r2s2 His / FLAG were then pooled, concentrated, and stored at -80°C.

[0313] 1.2. Expression and purification of human C1r2s2 tetramer The sequences used for expression and purification are 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 has the R463Q S654A mutation (SEQ ID NO: 4). For expression of recombinant human C1r2s2 tetramer, human C1s and human C1r were transiently co-expressed using the FreeStyle 293-F cell line (Thermo Fisher, Carlsbad, CA, USA). Culture supernatant 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 tetramer were collected, concentrated, and then applied to a Superdex 200 gel filtration column (GE healthcare) equilibrated with 1x TBS, 2 mM CaCl2 buffer. Fractions containing recombinant human C1r2s2 tetramer were then pooled, concentrated, and stored at -80°C.

[0314] Example 2 2.1 Preparation of Cynomolgus Monkey Fc gamma receptor (Cynomolgus Monkey FcγR) Gene constructs of the extracellular domain of cynomolgus monkey FcγR were obtained by cDNA cloning of each cynomolgus monkey FcγR using methods known to those skilled in the art. The sequences of FcγR used for expression and purification are shown in the sequence listing: (SEQ ID NO: 5 for cynomolgus monkey FcγRIa, SEQ ID NO: 6 for cynomolgus monkey FcγRIIa1, SEQ ID NO: 7 for cynomolgus monkey FcγRIIa2, SEQ ID NO: 8 for cynomolgus monkey FcγRIIa3, SEQ ID NO: 9 for cynomolgus monkey FcγRIIb, SEQ ID NO: 10 for cynomolgus monkey FcγRIIIa(R), SEQ ID NO: 11 for cynomolgus monkey FcγRIIIa(S). A gene encoding a His tag was then added to the 3' end of each gene. The resulting genes were inserted into an expression vector designed for mammalian cell expression. This expression vector was introduced into human fetal kidney cell-derived FreeStyle293 cells (Invitrogen) to express the target protein. After culturing, the obtained culture supernatant was filtered and purified in principle by the following four steps. The first step was cation exchange chromatography using SP Sepharose FF, the second step was affinity chromatography for His tag (HisTrap HP), the third step was gel filtration column chromatography (Superdex200), and the fourth step was sterile filtration. The absorbance of the purified protein at 280 nm was measured using a spectrophotometer, and the concentration of the purified protein was determined using the extinction coefficient calculated by the PACE method (Protein Science 4:2411-2423 (1995)).

[0315] 2.2 Preparation of fetal cynomolgus monkey Fc receptor (cynomolgus monkey FcRn) Cynomolgus monkey FcRn complexed with β2-microglobulin (β2m) was prepared. The sequences of FcRn and β2m used for expression and purification are shown in the sequence listing: (SEQ ID NO: 27 for cynomolgus monkey FcRn, SEQ ID NO: 28 for β2m). The constructed plasmids for cynomolgus monkey FcRn and β2m were introduced into cells of human embryonic kidney carcinoma-derived cell line HEK293H (Invitrogen) by lipofection using 10% fetal bovine serum (Invitrogen). The resulting culture supernatant was collected, and FcRn was further purified by purifying using IgG Sepharose 6 Fast Flow (Amersham Biosciences) followed by Q Sepharose High Performance (GE Healthcare). After all the above purification procedures, the protein was sterile filtered.

[0316] Example 3 Preparation of anti-C1s antibody 3.1 Construction of COS0098pHv1 and preparation of COS0098pHv1-SG1077R and COS0098pHv1-SG1 Humanization of the variable regions of anti-C1s antibody COS0098bb (VH, SEQ ID NO: 12; VL, SEQ ID NO: 13, as described in PCT / JP2018 / 042054) was performed to reduce the potential immunogenicity of this antibody. The complementarity determining regions (CDRs) of the anti-C1s rabbit antibody were grafted onto a homologous human antibody framework (FR) using conventional CDR grafting techniques (Nature 321:522-525 (1986)). A gene encoding the humanized VH was synthesized and linked to human IgG1 CH (SG1, SEQ ID NO: 14), modified human IgG1 CH, such as SG1077R (SEQ ID NO: 15) and SG1148 (SEQ ID NO: 16). A gene encoding the humanized VL was synthesized and linked to human CL (SK1, SEQ ID NO: 26) and modified human CL (K0MC, SEQ ID NO: 17). Each of the linked sequences was cloned into an expression vector.

[0317] To identify mutations and combinations of mutations that improve the binding properties of this lead antibody, many mutations and combinations of mutations were examined. Next, multiple mutations were introduced into the humanized variable region to enhance the binding affinity to C1s (human C1s or C1r2s2 or cynomolgus C1s2s2 His / Flag) at neutral pH or to decrease the binding affinity to C1s at acidic pH. Thus, 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 generated from COS0098bb. COS0098pHv1-SG1077R (for heavy chain, VH of COS0098pHv1, SEQ ID NO:18 combined with SG1077R, SEQ ID NO:15, and for light chain, VL of COS0098pHv1, SEQ ID NO:19 combined with SK1, SEQ ID NO:26) was used in cynomolgus monkeys in in vivo PK studies. COS0098pHv1-SG1 (for heavy chain, VH of COS0098pHv1, SEQ ID NO:18 combined with SG1, SEQ ID NO:14, and for light chain, VL of COS0098pHv1, SEQ ID NO:19 combined with SK1, SEQ ID NO:26) was used as a control antibody in cynomolgus monkey FcγR binding assays.

[0318] Antibodies were expressed in HEK293 cells co-transfected with a mixture of heavy and light chain expression vectors and purified by protein A. Further gel filtration was performed if necessary.

[0319] 3.2 Preparation of IPN009VH2VK3-SG4GK The polynucleotides of the heavy and light chain variable regions of the anti-C1s antibody, IPN009VH2 (SEQ ID NO: 30) and IPN009VK3 (SEQ ID NO: 31) (described in WO2016164358), were synthesized by GenScript Inc. The heavy and light chain variable regions were cloned into an expression vector containing the heavy chain constant region SG4GK (SEQ ID NO: 29) and the light chain constant region SK1 (SEQ ID NO: 26), respectively. The anti-C1s antibody C1_IPN009VH2-SG4GK / IPN009VK3-SK1 (IPN009VH2 / IPN009VK3-SG4GK) was transiently expressed in FreeStyle 293-F cells transfected with the vector by 293fectin (Life technologies) according to the manufacturer's instructions. Recombinant antibodies were purified using Protein A (GE Healthcare) and eluted in D-PBS, Tris-buffered saline (TBS), or His buffer (20 mM histidine, 150 mM NaCl, pH 6.0). Size exclusion chromatography was further performed to remove high and / or low molecular weight components, if necessary.

[0320] For large-scale preparation of anti-C1s antibody IPN009VH2-SG4GK / IPN009VK3-SK1 (also called IPN009VH2VK3-SG4GK) for cynomolgus monkey PK study, the antibody was transiently expressed in Expi293F cells (Life technologies) according to the manufacturer's instructions. The recombinant antibody was purified using ToyoScreen AF-rProteinA HC-650F (Tosoh Bioscience) and eluted with 50 mM acetic acid. The eluted antibody was neutralized with 1.5M Tris HCl / 1M arginine HCl buffer. To remove high and / or low molecular weight components, size exclusion chromatography was performed in P1 buffer (20 mM His, 150 mM Arg, 162.1 mM Asp, pH 6).

[0321] Example 4 Evaluation of the binding properties of anti-C1s antibodies 4.1. Evaluation of pH-dependency and cross-reactivity tests Pharmacokinetic parameters of anti-C1s antibodies against recombinant human C1s (described in PCT / JP2018 / 042054), human C1r2s2, and cynomolgus C1r2s2 His / Flag (also referred to as cynomolgus C1r2s2 in tables and figures) were evaluated at 37° C., pH 7.4, and pH 5.8 using a BIACORE® T200 instrument (GE Healthcare) or a BIACORE 4000 (GE Healthcare). ProA / G (Pierce) was immobilized on a CM4 sensor chip using an amine coupling kit (GE Healthcare) according to the settings recommended by GE Healthcare. Antibodies and analytes were diluted in their 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, 0.05 w / v% NaN3). Each antibody was captured on the sensor surface by ProA / G. The amount of antibody captured was typically 100-200 resonance units (RU). Recombinant C1s at concentrations of 25 and 50 nM was then injected, followed by dissociation at pH 7.4 and pH 5.8. The surface was regenerated with 10 mM glycine-HCl, pH 1.5. Pharmacokinetic parameters at neutral and acidic pH conditions were determined by fitting the sensorgrams to a 1:1 binding model using BIACORE® T200 Evaluation Software, version 2.0 (GE Healthcare) or BIACORE 4000 Evaluation Software. (pH 5.8 in Table 2) *The dissociation rates at pH 5.8 (after dissociation at pH 7.4, shown in (a)) were determined by processing and fitting the data using Scrubber 2.0 (BioLogic Software) curve fitting software. The sensorgrams of all antibodies at an antigen concentration of 50 nM are shown in Figures 1 and 2. The association rates (ka), dissociation rates (kd), binding affinities (KD) of these antibodies at pH 7.4 and pH 5.8 conditions, as well as the pH 5.8 dissociation rates were calculated. * The dissociation rates (kd) under acidic conditions are shown in Table 2. Some pharmacokinetic parameters of COS0098pHv1 under acidic conditions could not be calculated accurately due to weak binding reactivity. 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, whereas COS0098pHv1 (VH, SEQ ID NO:18 and VL, SEQ ID NO:19) exhibited a faster dissociation rate at pH 5.8 than at pH 7.4.

[0322] BIACORE® kinetic analysis was also performed to observe the cross-reactivity of anti-C1s antibodies to human C1r2s2 and cynomolgus C1r2s2 (cynomolgus C1r2s2 His / Flag). The binding kinetics and affinity at pH 7.4 are shown in Table 2. All anti-C1s antibodies showed comparable KDs to human C1r2s2 and cynomolgus C1r2s2.

[0323] [Table 2]

[0324] 4.2. Evaluation of antibody binding activity to cynomolgus monkey FcγR using Biacore Analysis of the interaction between the antibody prepared as described above and FcγR was performed using BIACORE® T200 (GE Healthcare). 50 mM phosphate buffer (pH 7.4) containing 150 mM sodium chloride and 0.05 w / v% Tween-20 was used as the running buffer, and the measurement temperature was set to 25 degrees. A chip was used that was prepared by immobilizing Protein L (BioVision) on a Series S sensor chip CM4 (GE Healthcare) by the amine coupling method. After capturing 500 or 1000 resonance units (RU) of the antibody of interest on the chip, it was allowed to interact with FcγR diluted in the running buffer, and their binding amount to the antibody was measured. Cynomolgus monkey FcγRIa was diluted to 8 nM, and the others were diluted to 1000 nM. The antibody captured on the chip was then washed with 10 mM glycine-HCl, pH 1.5, and the chip was regenerated and used repeatedly. Using the biacore evaluation software, the amount of FcγR bound was divided by the amount of each antibody bound to the capture antibody to obtain the binding activity, and these values ​​were compared. Furthermore, the relative values ​​of the binding activity of COS0098pHv1-SG1077R to each FcγR were calculated compared to COS0098pHv1-SG1 (an antibody having the sequence of natural human IgG1 at positions 234-239, 265-271, 295, 296, 298, 300, and 324-337 according to EU numbering).

[0325] Tables 3 and 4 show the binding activity values ​​of the antibodies and the relative amount of COS0098pHv1-SG1077R to COS0098pHv1-SG1. The binding activity values ​​of COS0098pHv1-SG1077R to all types of cynomolgus monkey FcγRIIa and FcγRIIb were higher than that of COS0098pHv1-SG1. Compared with COS0098pHv1-SG1, the binding activity of COS0098pHv1-SG1077R increased 2.86-fold to FcγRIIb and increased 2.23-3.96-fold to all types of cynomolgus monkey FcγRIIa.

[0326] [Table 3]

[0327] [Table 4]

[0328] 4.3 Evaluation of binding activity to cynomolgus monkey FcRn using Biacore The interaction between each antibody prepared as above and FcRn was analyzed using BIACORE (registered trademark) T200 (GE Healthcare). A buffer solution containing 150 mM sodium chloride, 0.05 w / v% Tween-20, and 50 mM phosphate buffer (pH 6.0) was used as the running buffer, and the measurement temperature was set to 25 degrees. A Series S sensor chip CM4 (GE Healthcare) with Protein L (BioVision) immobilized by the amine coupling method was used. After capturing 400 resonance units (RU) of the antibody of interest on the chip through Protein L, it was allowed to interact with FcRn diluted in the running buffer, and then its KD value was calculated. COS0098pHv1-SG1 was evaluated using 0, 250, 500, 1000, 2000, and 4000 nM of cynomolgus monkey FcRn, and COS0098pHv1-SG1077R was evaluated using 0, 15, 30, 60, 120, and 240 nM of cynomolgus monkey FcRn. The chips capturing each antibody were then washed with 10 mM glycine-HCl, pH 1.5, regenerated, and used repeatedly. Based on the steady-state analysis, the equilibrium dissociation constant (KD(M)) for each antibody against cynomolgus monkey FcRn was calculated. Each parameter was calculated using Biacore T200 evaluation software (GE Healthcare).

[0329] At pH 6.0, the KD value for the binding of COS0098pHv1-SG1 to cynomolgus monkey FcRn was 1.25 μmol / L, and the KD value for the binding of COS0098pHv1-SG1077R to cynomolgus monkey FcRn was 70.0 nmol / L.

[0330] Example 5 5.1 Cynomolgus monkey PK study Measurement of total cynomolgus monkey C1s and C1q concentrations in monkey plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry (LC / ESI-MS / MS)

[0331] method Measurement of total cynomolgus C1s and C1q concentrations in cynomolgus monkey plasma by high-performance liquid chromatography-electrospray tandem mass spectrometry (LC / ESI-MS / MS)

[0332] [Sample preparation] Total cynomolgus C1s concentrations in plasma were measured by LC / ESI-MS / MS. Calibration standards were prepared by mixing and diluting a defined amount of cynomolgus C1s in mouse plasma to give cynomolgus C1s concentrations of 0.954, 1.91, 3.82, 7.63, 15.3, 30.5, and 61.1 μg / mL. Cynomolgus plasma samples were diluted with mouse plasma. 2 μL of calibration standard or diluted plasma sample was mixed with 26.5 μL of mixed reagent (7.5 mol / L urea / 100 mmol / L dithiothreitol / 10 μg / mL lysozyme (chicken egg white) / 300 μg / mL human C1s=20 / 2 / 2.5 / 2) in 50 mmol / L ammonium bicarbonate and incubated at 56°C for 45 min. Then, 2 μL of 500 mmol / L iodoacetamide was added and incubated for 30 min in the dark at 37° C. 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 residual trypsin. 50 μL of the digested sample was subjected to analysis by LC / ESI-MS / MS.

[0333] The concentration of cynomolgus monkey C1q in plasma was measured by LC / ESI-MS / MS. Calibration standards were prepared by mixing and diluting a defined amount of cynomolgus monkey C1q in human plasma to give 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 with human plasma. 2 μL of calibration standards and diluted plasma samples were mixed with 40 μg of anti-human C1q antibody (Cell Sciences) in 1% bovine serum albumin and the administered anti-C1s antibody, respectively, for 1.5 hours at 25°C. Each sample was mixed with 50 μL of 10-fold diluted Protein A magnetic beads (Protenova) in PBS containing 0.05% Tween-20 for 1.5 hours at 25°C. After washing three times with PBS containing 0.05% Tween-20, the samples were mixed with 24.5 μL of mixed reagent (7.5 mol / L urea / 100 mmol / L dithiothreitol / 10 μg / mL lysozyme (chicken egg white) = 20 / 2 / 2.5) in 50 mmol / L ammonium bicarbonate 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 0.621 μg / mL sequencing grade modified trypsin (Promega) in 50 mmol / L ammonium bicarbonate was added and incubated at 37 °C overnight. Finally, 5 μL of 10% trifluoroacetic acid was added to inactivate residual trypsin. 50 μL of digested sample was subjected to analysis by LC / ESI-MS / MS.

[0334] [LC / ESI-MS / MS analysis] LC / ESI-MS / MS was performed using a Xevo TQ-S triple quadrupole instrument (Waters) equipped with a 2D I-class UPLC (Waters). The peptides LLEVPEAR (SEQ ID NO: 32), LLEVPEGR (SEQ ID NO: 33), and YQSVFTVAR (SEQ ID NO: 34), which are specific for cynomolgus C1s, human C1s, and cynomolgus C1s, respectively, were monitored by selected reaction monitoring (SRM). The SRM transitions were [M+2H]2+ (m / z 463.8~y6 1+ (m / z 700.3)), [M+2H] 2+ (m / z 456.8~y6 1+ (m / z 686.3)) and [M+2H] 2+ (m / z 535.8~y7 1+ (m / z 779.4). Each calibration curve was generated by weighted (1 / x2) linear regression with peak areas plotted against concentration. Concentrations in cynomolgus monkey plasma were calculated from the calibration curves using the analytical software Masslynx version 4.1 (Waters).

[0335] Evaluation of the pharmacokinetics of total C1s and C1q after administration of anti-C1s antibody in cynomolgus monkeys The in vivo pharmacokinetics of cynomolgus C1s and C1q was evaluated after administration of COS0098pHv1-SG1077R or IPN009VH2VK3-SG4GK to cynomolgus monkeys. Three male monkeys were assigned to each treatment group. On day 0, COS0098pHv1-SG1077R was intravenously injected into the monkeys at a dose of 10 mg / kg. Blood was collected 1 day before injection and 5 minutes, 2 hours, 8 hours, 1 day, 2 days, 4 days, 7 days, 14 days, 21 days, and 28 days after injection. IPN009VH2VK3-SG4GK was intravenously injected into the monkeys at a dose of 45 mg / kg once a week for a total of four doses. Blood was collected 1 day before injection, and 5 minutes, 2 hours, 8 hours, 1 day, 2 days, and 4 days after the first injection, the day immediately before and 24 hours after the second and third injections, and the day immediately before and 5 minutes, 2 hours, 8 hours, 1 day, 2 days, 4 days, and 7 days after the fourth injection. The dose settings of COS0098pHv1-SG1077R and IPN009VH2VK3-SG4GK were adjusted to be in excess of physiological plasma cynomolgus C1s and C1q concentrations immediately after injection. These blood samples were immediately centrifuged to separate plasma samples. At each sample collection time point, the plasma concentrations of cynomolgus C1s and C1q were measured by LC / ESI-MS / MS. The results are shown in Figures 3 and 4.

[0336] SG1077R contains mutations that enhance Fc gamma receptor binding. The plasma time-concentration profiles of cynomolgus C1s and C1q after IPN009VH2VK3-SG4GK injection were not significantly altered. On the other hand, the plasma concentration of cynomolgus C1s decreased to less than 30% of baseline after COS0098pHv1-SG1077R injection. Similarly, the plasma concentration of cynomolgus C1q also decreased to less than 20% of baseline after COS0098pHv1-SG1077R injection. In all samples, plasma C1q concentrations 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 indicates that COS0098pHv1-SG1077R has a strong ability to promote the removal of C1s and C1q. [Industrial Applicability]

[0337] The present invention provides antigen-binding molecules that contain specific amino acids that can bind to C1s and change its antigen-binding ability. Such antigen-binding molecules are expected to promote the removal of C1s and C1q from plasma.

Claims

1. An antigen-binding molecule that 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, 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, and an Fc region, the heavy chain variable region comprises 95% identity to the amino acid sequence of SEQ ID NO: 18, and the light chain variable region comprises 95% identity to the amino acid sequence of SEQ ID NO: 19; (i) the heavy chain variable region and / or the light chain variable region comprises at least one amino acid that can increase the KD(acidic pH range) / KD(neutral pH range), which is the ratio of the KD value of the antigen-binding molecule for C1s in the acidic pH range to the KD value of the antigen-binding molecule for C1s in the neutral pH range, compared to that of a first reference antibody comprising two heavy chain variable regions and two light chain variable regions, wherein the heavy chain variable region of the first reference antibody comprises the amino acid sequence of SEQ ID NO: 12, and the light chain variable region of the first reference antibody comprises the amino acid sequence of SEQ ID NO: 13; (ii) the Fc region comprises at least one amino acid that can enhance the binding ability of the antigen-binding molecule to FcRn in the acidic pH range compared to that of a second reference antibody comprising a paired Fc region, the Fc region of the second reference antibody comprising the amino acid sequence of SEQ ID NO: 14; and (iii) the Fc region comprises at least one amino acid that can enhance the binding ability of the antigen-binding molecule to an Fcγ receptor in the neutral pH range compared to that of a second reference antibody; Antigen-binding molecules.

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

3. The antigen-binding molecule according to claim 1 or 2, wherein the antigen-binding molecule comprises a constant region, the Fc region of the antigen-binding molecule is within the constant region, and the constant region comprises at least one amino acid capable of reducing the immunogenicity of the antigen-binding molecule.

4. The antigen-binding molecule according to any one of claims 1 to 3, wherein the Fc region of the antigen-binding molecule comprises at least one amino acid capable of reducing the binding activity to rheumatoid factor.

5. The variable region of (i) comprises the following amino acids: a histidine at position 27 in the heavy chain variable region; a proline at position 59 in the heavy chain variable region, and Histidine at position 96 in the light chain variable region (All numbers follow the Kabat numbering system) The antigen-binding molecule according to any one of claims 1 to 4, comprising at least one of:

6. The antigen-binding molecule according to any one of claims 1 to 5, wherein the Fc region of (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 according to the EU numbering system).

7. The Fc region of (iii) comprises the following amino acids: Tyrosine at position 234, Tryptophan at 235th position, Asparagine at position 236, Aspartic acid at position 238; Balin, ranked 250th; Isoleucine at position 264, Aspartic acid at position 268; Leucine at 295th place; Proline at position 307; Threonine at position 326, and Lysine at position 330 (All numbers follow the EU numbering system) The antigen-binding molecule according to any one of claims 1 to 6, comprising at least one of the following:

8. The Fc region of (iii) comprises the following amino acid (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 follow the EU numbering system) The antigen-binding molecule according to any one of claims 1 to 7, comprising:

9. The antigen-binding molecule of claim 2, wherein the at least one amino acid capable of improving stability is either or both of alanine at position 96 in the heavy chain variable region and glutamic acid at position 53 in the light chain variable region (both numbers 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 the binding activity to rheumatoid factor is either or both of arginine at position 438 and glutamic acid at position 440 (both numbers according to the EU numbering system).

12. The antigen-binding molecule according to any one of claims 1 to 11, wherein the heavy chain variable region and the light chain variable region comprise human-derived frameworks.

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