Formulations for anti-C1Q antibodies

Abstract

The present disclosure relates generally to pharmaceutical compositions comprising an anti-C1q antibody and methods of making and using such compositions. In certain embodiments, such compositions are high concentration anti-C1q antibody formulations that exhibit low viscosity and high stability, and thus can be conveniently delivered to subjects in need thereof to reduce discomfort.

Description

[0001] Related Applications

[0002] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 414,206, filed on October 7, 2022, which is hereby incorporated by reference in its entirety. Background Art

[0003] Therapeutic macromolecules (e.g., antibodies, antibody fragments and antibody derivatives) must be formulated in such a way that the molecules are not only suitable for application to patients, but also their stability is maintained during storage and subsequent use. For example, unless the solution is properly formulated, the therapeutic antibodies in the liquid solution are prone to degradation, aggregation or undesirable chemical modifications. The stability of the antibody in the liquid formulation depends not only on the type of excipient used in the formulation, but also on the amount and ratio of the excipient relative to each other. In addition, when preparing liquid antibody formulations, other considerations other than stability must be considered. Examples of such additional considerations include the viscosity of the solution, the antibody concentration that a given formulation can accommodate, and the visual quality or appearance of the formulation. Therefore, when preparing therapeutic antibodies, it is necessary to be very careful to obtain a formulation that maintains stability, contains sufficient concentrations of antibodies and has a suitable viscosity and enables the formulation to be easily applied to other properties of the patient.

[0004] Anti-CIq antibodies are one example of a therapeutically relevant macromolecule that requires an appropriate formulation.Anti-CIq antibodies are clinically useful for treating or preventing diseases associated with complement activation, such as autoimmune, inflammatory and neurodegenerative diseases, and cancer.

[0005] Although anti-C1q antibodies are known, there remains a need in the art for new pharmaceutical formulations comprising anti-C1q antibodies that are sufficiently stable and suitable for administration to patients. Summary of the invention

[0006] In certain aspects, provided herein are pharmaceutical compositions comprising anti-C1q antibodies, e.g., full-length antibodies, antibody Fab fragments, and / or antibody derivatives, and methods of using and preparing such pharmaceutical compositions. In certain embodiments, provided herein are pharmaceutical compositions comprising high concentration anti-C1q antibody solutions (whether full-length antibodies, antibody Fab fragments, and / or antibody derivatives) that exhibit both high dosage stability and low viscosity. Thus, such compositions are easily delivered, e.g., by intravitreal, subcutaneous, or intravenous injection to patients with diseases associated with the complement pathway. In addition, the high antibody concentration of the compositions provided herein combined with the low viscosity allows for the delivery of lower volumes of the composition to the patient through a smaller needle, thereby reducing patient discomfort.

[0007] Provided herein is a pharmaceutical composition comprising at least 75 mg / ml of an anti-C1q antibody. The pharmaceutical composition may further comprise 5 mM to 25 mM sodium citrate or sodium succinate; 3% to 7% trehalose; and / or 0.04% to 0.06% polysorbate 20 or polysorbate 80. The pH of the pharmaceutical composition may be 6.4-7.5.

[0008] In some embodiments, the antibody comprises a light chain variable domain comprising HVR-L1 having an amino acid sequence of SEQ ID NO: 5, HVR-L2 having an amino acid of SEQ ID NO: 6, and HVR-L3 having an amino acid of SEQ ID NO: 7. In some embodiments, the antibody comprises a heavy chain variable domain comprising HVR-H1 having an amino acid sequence of SEQ ID NO: 9, HVR-H2 having an amino acid of SEQ ID NO: 10, and HVR-H3 having an amino acid of SEQ ID NO: 11. The antibody may be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antibody derivative thereof, such as a one-armed antibody.

[0009] In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, and wherein the light chain variable domain comprises HVR-L1 having an amino acid sequence of SEQ ID NO: 5, HVR-L2 having amino acids of SEQ ID NO: 6, and HVR-L3 having amino acids of SEQ ID NO: 7. The light chain variable domain may comprise an amino acid sequence selected from SEQ ID NOs: 4 and 35-38. In some embodiments, the antibody comprises a heavy chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 8 and 31-34, and wherein the heavy chain variable domain comprises HVR-H1 having an amino acid sequence of SEQ ID NO: 9, HVR-H2 having amino acids of SEQ ID NO: 10, and HVR-H3 having amino acids of SEQ ID NO: 11. The heavy chain variable domain may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 31-34.

[0010] In some embodiments, the antibody is an antibody fragment, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a diabody or a single-chain antibody molecule. The antibody Fab fragment may preferably comprise a heavy chain Fab fragment of SEQ ID NO: 39. The antibody Fab fragment may preferably comprise a light chain Fab fragment of SEQ ID NO: 40.

[0011] In some embodiments, the pharmaceutical composition comprises at least 100 mg / ml, at least 125 mg / ml, at least 150 mg / ml, at least 175 mg / ml, at least 200 mg / ml, at least 225 mg / ml, or at least 250 mg / ml of anti-C1q antibody. The pharmaceutical composition may comprise 100 mg / ml to 125 mg / ml, 125 mg / ml to 150 mg / ml, 150 mg / ml to 175 mg / ml, 175 mg / ml to 200 mg / ml, or 200 mg / ml to 250 mg / ml of anti-C1q antibody. In some embodiments, the pharmaceutical composition comprises 75 mg / ml to 300 mg / ml, 100 mg / ml to 300 mg / ml, or 125 mg / ml to 250 mg / ml of anti-C1q antibody. In some embodiments, the pharmaceutical composition comprises about 75 mg / ml, 100 mg / ml, 125 mg / ml, about 150 mg / ml, about 175 mg / ml, about 200 mg / ml, 225 mg / ml, or about 250 mg / ml of the anti-C1q antibody Fab fragment.

[0012] In some embodiments, the pharmaceutical composition comprises about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM or about 25mM sodium citrate or sodium succinate. The pharmaceutical composition may comprise 5mM to 10mM, 10mM to 15mM, 15mM to 20mM or 20mM to 25mM sodium citrate or sodium succinate.

[0013] In some embodiments, the pharmaceutical composition is hypertonic or isotonic. In some embodiments, the pharmaceutical composition comprises about 3%, 4%, 5%, 6% or 7% trehalose. The pharmaceutical composition may comprise 3%-4%, 4%-5%, 5%-6% or 6%-7% trehalose. In some embodiments, the pharmaceutical composition comprises about 0.04%, 0.05% or 0.06% polysorbate 20 or polysorbate 80. The pharmaceutical composition may comprise 0.04% to 0.05% or 0.05% to 0.06% polysorbate 20 or polysorbate 80. In some embodiments, the pH of the pharmaceutical composition is about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4 or about 7.5. The pH of the pharmaceutical composition can be in the range of 6.0-6.5 or 6.5-7.0 or 7.0-7.5. In some embodiments, the pharmaceutical composition further comprises 30mM to 50mM NaCl. The pharmaceutical composition can comprise about 30mM, about 35mM, about 40mM, about 45mM or about 50mM NaCl. The pharmaceutical composition can comprise 30mM to 35mM, 35mM to 40mM, 40mM to 45mM or 45mM to 50mM NaCl.

[0014] In some embodiments, the viscosity of the pharmaceutical composition does not exceed 10 cP at 25° C., such as about 1 cP, about 2 cP, about 3 cP, about 4 cP, about 5 cP, about 6 cP, about 7 cP, about 8 cP, about 9 cP, or about 10 cP at 25° C. The viscosity of the pharmaceutical composition may be 1 cp to 5 cp or 5 cp to 10 cp at 25° C.

[0015] In some embodiments, the pharmaceutical composition is stable for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 20 weeks, at least 21 weeks, at least 22 weeks, at least 23 weeks, or at least 24 weeks. The pharmaceutical composition can be stable for at least 1 year, at least 1.5 years, or at least two years. The pharmaceutical composition can be stable at a temperature of -20°C to 25°C, such as about -20°C, about -15°C, about -10°C, about -5°C, about 0°C, about 4°C, about 5°C, about 10°C, about 15°C, about 20°C, or about 25°C. In some embodiments, the pharmaceutical composition is stable at a temperature greater than or equal to 25°C, such as between about 25°C to about 40°C (eg, about 25°C, about 30°C, about 35°C, or about 40°C).

[0016] In some embodiments, the pharmaceutical composition is stable after at least one, at least two, at least three, at least four, or at least five freeze / thaw cycles. In some embodiments, the pharmaceutical composition is stable after continuous stirring for at least one day, at least two days, or at least three days.

[0017] In some embodiments, the pharmaceutical composition is suitable for intravitreal or intraocular injection. The pharmaceutical composition can include 5mM to 20mM sodium citrate or sodium succinate, such as about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM or about 20mM sodium citrate or sodium succinate.

[0018] In some embodiments, the pharmaceutical composition is contained in a syringe. In some embodiments, the pharmaceutical composition is pre-filled in a syringe. The syringe may have a fill volume of no more than 300 microliters. In some embodiments, the syringe is configured to deliver a volume of 25 microliters-100 microliters. In some embodiments, the syringe is silicon-free and / or has a low level of microparticles. In some embodiments, the pharmaceutical composition is silicon-free and / or has a low level of microparticles. In some embodiments, the pre-filled syringe has less than fifty particles of 10μm particles per milliliter and / or less than five particles of 25μm particles per milliliter. In some embodiments, the pharmaceutical composition has less than fifty particles of 10μm particles per milliliter and / or less than five particles of 25μm particles per milliliter. In some embodiments, the pre-filled syringe complies with USP <789> In some embodiments, the pharmaceutical composition complies with USP <789> Limit value.

[0019] In some embodiments, the pharmaceutical composition is prepared for subcutaneous injection. The pharmaceutical composition can be contained in a syringe, an automatic injector, or a pen. The syringe, automatic injector, or pen can have a fill volume of no more than 10 ml.

[0020] In some embodiments, the pharmaceutical composition is prepared for intravenous injection.The pharmaceutical composition can be contained in an intravenous solution bag.

[0021] In one aspect, disclosed herein is a syringe comprising a pharmaceutical composition disclosed herein. In some embodiments, the syringe has a delivery volume of no more than 10 ml. In some embodiments, the syringe is an automatic reusable fixed dose pen. Alternatively, the syringe can be an automatic reusable variable dose pen. In some embodiments, the syringe is an automatic disposable fixed dose auto-injector. The syringe can have a delivery volume of no more than 300 microliters. The syringe can be a syringe.

[0022] In another aspect, described herein is a pre-filled syringe comprising a pharmaceutical composition described herein. In some embodiments, the pre-filled syringe has a delivery volume of no more than 300 microliters. In some embodiments, the pre-filled syringe is silicon-free and / or has a low level of microparticles. In some embodiments, the pharmaceutical composition is silicon-free and / or has a low level of microparticles. In some embodiments, the pre-filled syringe has less than fifty particles per milliliter of 10 μm particles and / or less than five particles per milliliter of 25 μm particles. In some embodiments, the pre-filled syringe complies with USP <789> In some embodiments, the pharmaceutical composition complies with USP <789> Limit value.

[0023] In yet another aspect, a method of inhibiting synaptic loss is described herein. The method may include administering a pharmaceutical composition described herein to a patient suffering from adverse synaptic loss. In some embodiments, the patient suffers from synaptic loss due to a neurodegenerative disorder, a central nervous system disorder, or a peripheral nervous system disorder. The neurodegenerative disorder may be Guillain Barre Syndrome (GBS), amyotrophic lateral sclerosis (ALS), or Huntington's Disease (HD).

[0024] In one aspect, disclosed herein is a method for treating or preventing a disease associated with complement activation in an individual in need of such treatment. The method may include administering a pharmaceutical composition as described herein. The disease associated with complement activation may be a neurodegenerative disorder, which may be associated with synaptic loss or neural connection loss, associated with synaptic loss dependent on complement receptor 3 (CR3) / C3 or complement receptor CR1, associated with pathological activity-dependent synaptic pruning, or associated with synaptic phagocytosis of microglia. In some embodiments, the neurodegenerative disorder is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, ophthalmic disorders, glaucoma, myotonic dystrophy, Guillain-Barre syndrome (GBS), myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, traumatic brain injury (TBI), epilepsy or Huntington's disease (HD). In some embodiments, the neurodegenerative disorder is amyotrophic lateral sclerosis (ALS).In some embodiments, the neurodegenerative disorder is Huntington's disease.

[0025] In some embodiments, the disease associated with complement activation is an inflammatory disease, an autoimmune disease, a complement-associated eye disease, or a metabolic disorder. The inflammatory disease, autoimmune disease, complement-related eye disease or metabolic disorder can be selected from diabetes, obesity, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), distal tissue damage after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and induced glomerulonephritis and vasculitis, cardiopulmonary bypass, cardiac arrest-induced coronary endothelial dysfunction, type II membranous proliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, glaucoma, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degeneration, age-related macular degeneration (AMD), geographic atrophy, choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, intraocular neovascularization, diabetic macular edema, pathological myopia, von Hippel-Lindau disease (von Hippel-Lindau disease) disease, ocular histoplasmosis, neuromyelitis optica (NMO), central retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, ANCA vasculitis, Purtscher retinopathy, Sjogren's dry eye disease, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary disease (COPD), asthma, aspiration pneumonia, multiple sclerosis, multifocal motor neuropathy (MMN), Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis.

[0026] The disease associated with complement activation can be an autoimmune disease selected from the group consisting of multifocal motor neuropathy (MMN), type 1 diabetes, Hashimoto's thyroiditis, Addison's disease, celiac disease, Crohn's disease, pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolytic anemia, tumor-associated syndrome, vasculitic diseases, hypocomplementemic urticarial vasculitis (HUV), polymyalgia rheumatica, temporal arteritis, Wegener's gran ulomatosis, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid or myositis.

[0027] The disease associated with complement activation can be a blood disorder selected from the group consisting of cold agglutinin hemolytic anemia (cold agglutinin disease), cold antibody hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, neonatal alloimmune thrombocytopenia, red blood cell isotype induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, systemic lupus erythematosus (SLE), glomerulonephritis, antiphospholipid antibody syndrome (APS), infection or drug-induced hematological disorders. DETAILED DESCRIPTION

[0028] Overview

[0029] Provided herein is a novel pharmaceutical composition comprising an antibody, antibody fragment or antibody derivative capable of binding to C1q, particularly an antibody Fab fragment, and methods for preparing and using such pharmaceutical compositions. Also provided herein is the use of the pharmaceutical composition for preventing, reducing the risk of development or treating diseases associated with complement activation.

[0030] definition

[0031] As used herein in the specification, "a" or "an" may mean one or more. As used herein in one or more claims, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more than one. For example, a reference to an "antibody" is a reference to one to more than one antibodies. As used herein, "another" may refer to at least a second or more than one.

[0032] The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The term "antibody" is used in the broadest sense herein and specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed by at least two complete antibodies, antibody fragments (as long as they exhibit biological activity), and antibody derivatives. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two % identical light chains (L) and two % identical heavy chains (H). V H and V L Paired together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds), Appl eton & Lange, Norwalk, CT, 1994, page 71 and chapter 6. The L chain from any vertebrate species can be divided into one of two distinct types based on the amino acid sequence of its constant domain, called kappa (" κ ") and lambda (" λ "). Depending on the amino acid sequence of the constant domain of its heavy chain (CH), immunoglobulins can be divided into different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and their heavy chains are named alpha (" α "), delta (" δ "), Epsilon (" ε "), gamma (" γ ") and mu (" μ "). Based on relatively minor differences in CH sequence and function, the γ and α classes are further divided into subclasses (isotypes), e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known and generally described in, e.g., Abbas et al., Cellular and Molecular Immunology, 4th edition. (WB Saunders Co., 2000).

[0033] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy chain or light chain of an antibody. The variable domains of the heavy chain and light chain can be referred to as "V H ” and “V L ". These domains are usually the most variable part of the antibody (relative to other antibodies of the same class) and contain the antigen binding site. The variable domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, the variability is not evenly distributed across the entire span of the variable domain. In contrast, it is concentrated in three segments called hypervariable regions (HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions, which mostly adopt a β-sheet configuration, connected by three HVRs, which form loops connecting the β-sheet structure and sometimes form part of the β-sheet structure. The HVRs in each chain are tightly bound together by the FR region and, together with the HVRs from the other chain, help form the antigen binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions, such as the participation of the antibody in antibody-dependent cellular toxicity.

[0034] As used herein, the term "CDR" or "complementarity determining region" refers to non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996) describe CDRs, where the definitions include overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to the CDRs of an antibody or a grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein. As used herein, the terms "CDR-L1", "CDR-L2" and "CDR-L3" refer to the first, second and third CDRs in the light chain variable region, respectively. As used herein, the terms "CDR-H1", "CDR-H2" and "CDR-H3" refer to the first, second and third CDRs in the heavy chain variable region, respectively. As used herein, the terms "CDR-1", "CDR-2" and "CDR-3" refer to the first, second and third CDRs in either chain variable region, respectively.

[0035] Many HVR and CDR descriptions are being used and are included in this article. As the HVR of Kabat complementary determining region (CDR) is based on the variability of sequence, and is the most commonly used (Kabat et al., supra). Chothia refers to the position of the structural loop (Chothia and Lesk J.Mol.Biol.196:901-917 (1987)). AbM HVR represents a compromise between Kabat CDR and Chothia structural loops, and is used by the AbM antibody modeling software of Oxford Molecular. "Contact" HVR is based on the analysis of available complex crystal structures. The residues from each of these HV R are as follows.

[0036]

[0037] HVRs may include the following "extended HVRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, 26-35 (H1), 50-65 or 49-65 (preferred embodiments) (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these extended HVR definitions, the variable domain residues are numbered according to Kabat et al., supra.

[0038] When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., Kabat et al., EU index reported in the supra). "EU index as in Kabat" refers to the residue numbering of human IgG1EU antibodies. Unless otherwise specified herein, reference to residue numbering in antibody variable domains refers to residue numbering by the Kabat numbering system. Unless otherwise specified herein, reference to residue numbering in antibody constant domains refers to residue numbering by the EU numbering system (see, e.g., U.S. Patent Publication No. 2010-280227).

[0039] As used in the present specification, the expression "C1q antibody" should be interpreted similarly to "anti-C1q antibody" and means an antibody capable of binding to C1q. In one embodiment of the present application, the epitope of the antibody is located in the extracellular domain of human C1q.

[0040] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies in the population are identical, except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific (directed at a single antigenic site). In contrast to polyclonal antibody preparations that typically include different antibodies directed at different determinants (epitopes), each monoclonal antibody is directed at a single determinant on the antigen.

[0041] An "antibody fragment" or "antigen-binding fragment" or "functional fragment" of an antibody comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of an intact antibody, or the F region of an antibody that retains or has modified FcR binding ability. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; and linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)). Other examples of antibody fragments include antibody derivatives, such as single-chain antibody molecules, monovalent antibodies, and multispecific antibodies formed from antibody fragments.

[0042] An "antibody derivative" is any construct comprising the antigen-binding region of an antibody. Examples of antibody derivatives include single-chain antibody molecules, one-armed antibodies, antibodies with a single antigen-binding arm, monovalent antibodies, and multispecific antibodies formed from antibody fragments.

[0043] The term "single-arm antibody" herein is used to cover antibodies comprising a single antigen binding arm. A single-arm antibody may comprise an antigen binding arm and an Fc region, wherein the single antigen binding arm comprises a light chain variable domain and a heavy chain variable domain; and the Fc region comprises a complex of a first Fc polypeptide and a second Fc polypeptide. In some embodiments, one (but not both) of the Fc polypeptides is an N-terminally truncated heavy chain. In some embodiments, the N-terminally truncated heavy chain lacks a heavy chain variable domain, and preferably the N-terminally truncated heavy chain lacks a heavy chain constant domain 1. In some embodiments, the antibody may be a bivalent antibody, wherein one arm binds C1q and the other arm binds a different antigen. Depending on the other antigen, this antibody will not cross-link and activate C1q. A single-arm antibody may be an antibody with a single antigen binding arm. As used herein, "antibody with a single antigen binding arm" means an antibody comprising a single antigen binding arm and an Fc region, wherein the antigen binding arm comprises a light chain variable domain and a heavy chain variable domain. In some embodiments, the antibody further comprises an inactive antigen binding arm that is incapable of binding to an antigen, or the antibody comprises an arm that binds to a different antigen.In some embodiments, the Fc region comprises a complex of a first Fc polypeptide and a second Fc polypeptide.

[0044] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a name that reflects the ability to crystallize readily. The Fab fragment consists of an intact L chain and the variable region domain (V H ) and the first constant domain of a heavy chain (C H1) Composition. Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment that is roughly equivalent to two disulfide-linked Fab fragments with different antigen binding activities and is still able to cross-link antigen. The Fab' fragment differs from the Fab fragment in that the C H The carboxyl terminus of the constant domain has several additional residues, including one or more cysteines from the antibody hinge region. Fab' in which the cysteine ​​residues of the constant domains have free thiol groups is referred to herein as Fab'-SH. F(ab')2 antibody fragments were initially produced in the form of Fab' fragment pairs that had hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

[0045] The Fc fragment comprises the carboxyl terminal portions of two H chains held together by disulfides. The effector functions of an antibody are determined by the sequence of the Fc region, which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0046] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be different, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (according to the EU numbering system, residue 447) in the Fc region can be removed, for example, during production or purification of antibodies or by recombinant engineering of the nucleic acid encoding the heavy chain of an antibody. Therefore, the composition of a complete antibody can include an antibody population in which all K447 residues are removed, an antibody population in which no K447 residue is removed, and an antibody population with and without an antibody mixture containing K447 residues. The native sequence Fc region suitable for antibodies of the present disclosure includes human IgG1, IgG2, IgG3, and IgG4.

[0047] A "native sequence Fc region" comprises an amino acid sequence that is % identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0048] A "variant Fc region" comprises an amino acid sequence that is distinguished from a native sequence Fc region amino acid sequence by at least one amino acid modification (preferably one or more amino acid substitutions). Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, for example, there are about one to about ten amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide, preferably about one to about five amino acid substitutions. The variant Fc region herein will preferably have at least about 80% homology with the native sequence Fc region and / or with the Fc region of a parent polypeptide, most preferably at least about 90% homology therewith, and more preferably at least about 95% homology therewith.

[0049] As used herein, "chimeric antibody" refers to an antibody (immunoglobulin) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as long as it exhibits the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). The chimeric antibodies of interest herein include Antibodies wherein the antigen binding region of the antibody is derived from an antibody produced by, for example, immunizing a macaque with an antigen of interest. As used herein, "humanized antibodies" are a subset of "chimeric antibodies"

[0050] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing minimal sequences derived from non-human immunoglobulins. In some embodiments, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the HVR of the receptor are replaced by residues from the HVR of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) with the desired specificity, affinity, and / or ability. In some cases, the FR residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may be included in a receptor antibody or in a residue not found in a donor antibody. These modifications may be performed to further improve the performance of the antibody, such as binding affinity. In general, a humanized antibody will include substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, but the FR region may include one or more individual FR residue substitutions, which may improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is usually no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0051] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or an antibody prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). Also useful for preparing human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1): 86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled, such as an immunized xenogeneic mouse (refer to XENOMOUSE TM For human antibodies generated by human B cell hybridoma technology, see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584). For human antibodies generated by human B cell hybridoma technology, see, e.g., Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006).

[0052] As used herein, "acceptor human framework" is a framework comprising an amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise an amino acid sequence that is % identical thereto, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing 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. When pre-existing amino acid changes are present in VH, it is preferred that these changes occur only at three, two or one positions in positions 71H, 73H and 78H; for example, the amino acid residues at those positions may be 71A, 73T and / or 78A. In some embodiments, the VL acceptor human framework sequence is identical to the VL human immunoglobulin framework sequence or a human consensus framework sequence.

[0053] "Human consensus framework" is a framework that represents the amino acid residues that are most commonly present in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include that for VL, the subgroup may be a subgroup κI, κII, κIII or κIV such as Kabat et al., supra. In addition, for VH, the subgroup may be a subgroup I, subgroup II or subgroup III such as Kabat et al., supra.

[0054] "Amino acid modification" at a specific position refers to the substitution or deletion of a specific residue, or the insertion of at least one amino acid residue near a specific residue. Insertion "adjacent" a specific residue refers to insertion within one to two residues thereof. Insertion can be at the N-terminus or C-terminus of a specific residue. The preferred amino acid modification herein is substitution.

[0055] As used herein, "identity" means that at any particular position of the aligned sequences, the amino acid residues between the sequences are the same. As used herein, "similarity" indicates that at any particular position of the aligned sequences, the amino acid residues between the sequences are of similar type. For example, leucine can replace isoleucine or valine. Other amino acids that can often be substituted for each other include, but are not limited to:

[0056] - Phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

[0057] - lysine, arginine and histidine (amino acids with basic side chains);

[0058] - Aspartic acid and glutamic acid (amino acids with acidic side chains);

[0059] - Asparagine and glutamine (amino acids with amide side chains); and

[0060] - Cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0061] The degree of identity and similarity can be readily calculated. (See, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., M Stockton Press, New York, 1991).

[0062] As used herein, an "interaction" between CIq and a second protein encompasses, but is not limited to, protein-protein interactions, physical interactions, chemical interactions, binding, covalent binding, and ionic binding. As used herein, an antibody "inhibits" an interaction between two proteins when the antibody disrupts, reduces, or completely eliminates the interaction between the two proteins. When an antibody or fragment thereof of the present disclosure binds to one of the two proteins, the antibody or fragment thereof "inhibits" an interaction between the two proteins.

[0063] As used herein, "percentage (%) of amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences refer to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after alignment of the sequences and introduction of gaps (if necessary) to achieve the maximum percentage of sequence identity, and without taking into account any conservative substitutions as part of the sequence identity. Alignment for determining percentage of amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full length of the sequences being compared.

[0064] The term "prevention" is art-recognized and well known in the art when used in conjunction with a condition, and includes administration of a composition that reduces the frequency of a medical condition, or delays the onset of a medical condition, symptoms of a medical condition, in a subject relative to a subject not receiving the composition. Thus, prevention of a disease associated with complement activation includes, for example, reducing the incidence of a disease associated with complement activation in a population of patients receiving prophylactic treatment relative to an untreated control population, and / or delaying the onset of a disease associated with complement activation in a treated population compared to an untreated control population, such as by a statistically and / or clinically significant amount.

[0065] As used herein, the term "specific recognition" or "specific binding" refers to a measurable and repeatable interaction, such as attraction or binding between a target and an antibody, which determines the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically or preferentially binds to a target or epitope is an antibody that binds to a target or epitope with greater affinity, avidity, greater ease, and / or greater duration than it binds to other targets or other epitopes of a target. It is also understood that, for example, an antibody (or portion) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but it may include) exclusive binding. An antibody that specifically binds to a target may have at least about 10 3 M -1 or 10 4 M -1 , sometimes about 10 5 M -1 or 10 6 M -1 , in other cases about 10 6 M -1 or 10 7 M -1 , about 10 8 M -1 Up to 10 9 M -1 or about 10 10 M -1 Up to 10 11 M -1or higher association constant. A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically immunoreact with a protein. For a description of immunoassay formats and conditions that can be used to determine specific immune responses, see, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0066] The term "subject" as used herein refers to a living mammal and can be used interchangeably with the term "patient". Examples of mammals include, but are not limited to, any member of the class of mammals: humans; non-human primates, such as chimpanzees and other apes and monkey species; livestock, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs and cats; laboratory animals, including rodents, such as rats, mice and guinea pigs, etc. The terms do not denote a particular age or sex.

[0067] The term "therapeutically effective amount" of a compound with respect to the subject treatment methods refers to an amount of the compound in a formulation that, when administered as part of a desired dosing regimen (to a mammal, preferably a human), alleviates symptoms, ameliorates symptoms, or slows the onset of a disease condition, according to clinically acceptable standards for the condition or disorder to be treated or for cosmetic purposes, e.g., at a reasonable benefit / risk ratio applicable to any drug treatment. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual.

[0068] As used herein, the terms "treating" or "treatment" include alleviating a disorder, arresting a disorder, or reversing the symptoms, clinical signs, or underlying pathology of a disorder, to stabilize or improve a subject's disorder, or to reduce the likelihood that a subject's disorder will worsen, as if the subject were not receiving treatment.

[0069] Antibody

[0070] All sequences mentioned in the present disclosure are incorporated by reference into U.S. Patent Application No. 14 / 933,517, U.S. Patent Application No. 14 / 890,811, U.S. Patent No. 8,877,197, U.S. Patent No. 9,708,394, U.S. Patent Application No. 15 / 360,549, U.S. Patent No. 9,562,106, U.S. Patent No. 10,450,382, U.S. Patent No. 10,457,745, International Patent Application No. PCT / US2018 / 022462, and the antibodies and related compositions disclosed in each of these patents are hereby incorporated by reference.

[0071] Antibodies can be prepared by using recombinant DNA engineering techniques. Such engineered versions include versions produced, for example, by inserting, deleting or changing the variable region of a natural antibody in the amino acid sequence of a natural antibody. Specific examples of this type include those engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from an antibody and the remainder of the variable region domain from a second antibody. The DNA encoding the antibody can be prepared by deleting all parts of the DNA encoding the antibody except the required part. The DNA encoding the chimeric antibody can be prepared by recombining DNA encoding people's constant region substantially or specifically and encoding the DNA of the variable region of the variable region sequence of mammals other than humans substantially or specifically. The DNA encoding the humanized antibody can be prepared by recombining the DNA encoding the constant region and the variable region except the complementary determining region (CDR) of the corresponding human antibody region substantially or specifically and encoding the DNA of the CDR of mammals other than humans substantially or specifically.

[0072] Suitable sources of DNA molecules encoding antibodies include cells expressing full-length antibodies, such as hybridomas. For example, antibodies can be isolated from host cells expressing expression vectors encoding the heavy and / or light chains of the antibodies.

[0073] Antibody fragments, including but not limited to Fab fragments and / or antibody derivatives, can also be prepared using recombinant DNA engineering techniques, which involve the manipulation and re-expression of DNA encoding the variable and constant regions of antibodies. Standard molecular biology techniques can be used to modify, add or delete more amino acids or domains as needed. The terms "variable" and "constant" regions as used herein still encompass any changes to the variable or constant regions. In some cases, by modifying the C H A stop codon was introduced immediately after the codon for the interchain cysteine ​​of 1, and PCR was used to generate antibody fragments such that C H The translation of the 1 domain is terminated at the interchain cysteine. Methods for designing suitable PCR primers are well known in the art, and antibody C H The sequence of the 1 domain is readily available. In some embodiments, a stop codon can be introduced using site-directed mutagenesis techniques.

[0074] The antibodies of the present disclosure can be derived from any antibody isotype ("class"), including, for example, IgG, IgM, IgA, IgD and IgE and subclasses thereof, including, for example, IgG1, IgG2, IgG3 and IgG4. In certain preferred embodiments, the heavy and light chains of the antibodies are from IgG. The heavy and / or light chains of the antibodies can be from mouse IgG or human IgG. In certain other preferred embodiments, the heavy and / or light chains of the antibodies are from human IgG1. In yet other preferred embodiments, the heavy and / or light chains of the antibodies are from human IgG4.

[0075] The antibodies disclosed herein can bind to and inhibit the biological activity of C1q, C1r or C1s. For example, (1) binding of C1q to autoantibodies, (2) binding of C1q to C1r, (3) binding of C1q to C1s, (4) binding of C1q to phosphatidylserine, (5) binding of C1q to pentraxin-3, (6) binding of C1q to C-reactive protein (CRP), (7) binding of C1q to globular C1q receptor (gC1qR), (8) binding of C1q to complement receptor 1 (CR1), (9) binding of C1q to β-amyloid protein, or (10) binding of C1q to calreticulin. In other embodiments, the biological activity of C1q is (1) activation of the classical complement activation pathway, (2) reduction in lysis and / or reduction in C3 deposition, (3) activation of antibodies and complement dependent cytotoxicity, (4) CH50 hemolysis, (5) reduction in erythrocyte lysis, (6) reduction in erythrocyte phagocytosis, (7) reduction in dendritic cell infiltration, (8) inhibition of complement-mediated erythrocyte lysis, (9) reduction in lymphocyte infiltration, (10) reduction in macrophage infiltration, (11) reduction in antibody deposition, (12) Reduction in neutrophil infiltration, (13) Reduction in platelet phagocytosis, (14) Reduction in platelet lysis, (15) Improvement in transplant graft survival, (16) Reduction in macrophage-mediated phagocytosis, (17) Reduction in autoantibody-mediated complement activation, (18) Reduction in red blood cell destruction caused by transfusion reactions, (19) Reduction in red blood cell lysis caused by alloantibodies, (20) Reduction in hemolysis caused by transfusion reactions, (21) Reduction in alloantibody-mediated platelet lysis, (22) Improvement in anemia , (23) reduction in eosinophilia, (24) reduction in C3 deposition on red blood cells (e.g., reduction in deposition of C3b, iC3b, etc. on RBCs), (25) reduction in C3 deposition on platelets (e.g., reduction in deposition of C3b, iC3b, etc. on platelets), (26) reduction in anaphylatoxin production, (27) reduction in autoantibody-mediated blister formation, (28) reduction in autoantibody-induced erythema, (29) reduction in red blood cell destruction caused by transfusion reactions, (30) reduction in blood transfusion reactions caused by transfusion reactions reduction in platelet lysis, (31) reduction in mast cell activation, (32) reduction in mast cell histamine release, (33) reduction in vascular permeability, (34) reduction in complement deposition on the transplant graft endothelium, (35) B cell antibody production, (36) dendritic cell maturation, (37) T cell proliferation, (38) cytokine production, (39) microglial activation, (40) Arthritis reaction, (41) reduction in anaphylatoxin production in the transplant graft endothelium, or (42) activation of complement receptor 3 (CR3 / C3) expressing cells.

[0076] In some embodiments, CH50 hemolysis includes human, mouse and / or rat CH50 hemolysis. In some embodiments, the antibody is capable of neutralizing at least about 50% to at least about 95% of CH50 hemolysis. In some embodiments, the antibody is capable of neutralizing 50%, 60%, 70%, 80%, 90% or 100% of CH50 hemolysis. The antibody is also capable of neutralizing at least 50% of CH50 hemolysis at a dose of less than 150 ng / ml, less than 100 ng / ml, less than 50 ng / ml or less than 20 ng / ml.

[0077] Other in vitro assays for measuring complement activity include ELISA assays for measuring cleavage products of complement components or complexes formed during complement activation. Complement activation through the classical pathway can be measured by tracking the levels of C4d and C4 in serum. Activation of the alternative pathway can be measured in an ELISA by assessing the levels of circulating Bb or C3bBbP complexes. In vitro antibody-mediated complement activation assays can also be used to assess inhibition of C3a production.

[0078] The antibodies of the present disclosure may be monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, human antibodies, chimeric antibodies, multispecific antibodies, antibody fragments thereof, or derivatives thereof, including one-armed antibodies. In some embodiments, the antibody is a humanized antibody.

[0079] The antibodies of the present disclosure may also be antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies or single-chain antibody molecules. In some embodiments, the antibody fragment is a Fab fragment.

[0080] In some embodiments, the antibody is a human monoclonal antibody that can be prepared, expressed, produced, or isolated by recombinant means, such as (a) an antibody isolated from an animal (e.g., mouse) that is transgenic or cross-chromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) an antibody isolated from a host cell transformed to express the antibody (e.g., from a transfectoma), (c) an antibody isolated from a recombinant, combinatorial human antibody library, and (d) an antibody prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when a transgenic animal for human Ig sequences is used, in vivo somatic mutagenesis), and thus the V H and V L The amino acid sequence of the region is such a sequence, which, although derived from human germline V H and V LSequences that are related to, but may not naturally exist within the human antibody germline repertoire in vivo.

[0081] In some embodiments, the antibody is a humanized and / or chimeric monoclonal antibody, which can be produced by immunizing rodents (e.g., mice, rats, hamsters, and guinea pigs) with (1) natural complement components (e.g., C1q) derived from enzymatic digestion of purified complement components from human plasma or serum, or (2) recombinant complement components expressed by eukaryotic or prokaryotic systems, or fragments thereof. Other animals can be used for immunization, such as non-human primates, transgenic mice expressing human immunoglobulins, and severe combined immunodeficient (SCID) mice transplanted with human B lymphocytes.

[0082] Polyclonal and monoclonal antibodies are naturally produced as immunoglobulin (Ig) molecules by the immune system in response to pathogens. The IgG1 molecule of about 150 kDa is the major form in human serum at a concentration of 8 mg / ml and is composed of two identical heavy chains of about 50 kDa and two identical light chains of about 25 kDa.

[0083] Hybridomas can be produced by conventional procedures by fusing B lymphocytes from immunized animals with myeloma cells. In addition, anti-C1q antibodies can be produced by screening recombinant single-chain Fv or Fab libraries from human B lymphocytes in a phage display system. The specificity of MAbs for human C1q can be tested by enzyme-linked immunosorbent assay (ELISA), Western immunoblotting or other immunochemical techniques.

[0084] The inhibitory activity of antibodies identified during the screening process on complement activation can be assessed by hemolytic assay using unsensitized rabbit or guinea pig RBCs for the alternative complement pathway or sensitized chicken or sheep RBCs for the classical complement pathway. Those hybridomas that exhibit inhibitory activity specific for the classical complement pathway are cloned by limiting dilution. Antibodies are purified by the above assays for characterization of specificity for human CIq.

[0085] Anti-complement C1q antibody

[0086] The anti-C1q antibodies disclosed herein are potent inhibitors of C1q and can be administered for any period of time to provide continued inhibition of C1q function and then optionally discontinued to allow restoration of normal C1q function when its activity may be important.

[0087] C1q is a 460 kDa polymeric protein consisting of 18 polypeptide chains (6 C1qA chains, 6 C1q B chains, and 6 C1q C chains). The C1r and C1s complement proteins bind to the C1q tail region to form the C1 complex (C1qr2s2).

[0088] The antibodies disclosed herein specifically recognize complement factor C1q and / or C1q in the C1 complex of the classical complement activation pathway. The binding complement factor can be derived from any organism with a complement system without limitation, including any mammalian organism, such as humans, mice, rats, rabbits, monkeys, dogs, cats, cows, horses, camels, sheep, goats or pigs.

[0089] As used herein, "C1 complex" refers to a protein complex that may include, but is not limited to, one C1q protein, two C1r proteins, and two C1s proteins (e.g., 2 s 2 ).

[0090] The anti-C1q antibodies disclosed herein can inhibit C1 complex formation.

[0091] As used herein, "complement factor C1q" refers to both the wild-type sequence and naturally occurring variant sequences.

[0092] A non-limiting example of a complement factor C1q recognized by the antibodies of the present disclosure is human C1q, comprising three polypeptide chains A, B and C:

[0093] C1q, chain A (homo sapiens), accession number protein

[0094] Database: NP_057075.1; GenBank ID: NM_015991:

[0095] >gi|7705753|ref|NP_057075.1|Component C1q

[0096] Subunit A precursor [Homo sapiens]

[0097] (SEQ ID NO:1)

[0098] MEGPRGWLVLCVLAISLASMVTEDLCCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAIRRN PPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA.

[0099] C1q, chain B (Homo sapiens), accession number protein

[0100] Database: NP_000482.3; GenBank ID: NM_000491.3:

[0101] >gi|87298828|ref|NP_000482.3|Component Clq

[0102] Subunit B precursor [Homo sapiens]

[0103] (SEQ ID NO:23)

[0104] MMMKIPWGSIPVLMLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSAT RTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA.

[0105] C1q, chain C (Homo sapiens), accession number protein

[0106] Database: NP_001107573.1; GenBank ID:

[0107] NM_001114101.1:

[0108] >gi|166235903|ref|NP_001107573.1|Component C1q

[0109] Subcomponent subunit C precursor [Homo sapiens]

[0110] (SEQ ID NO:24)

[0111] MDVGPPSSLPHLGLKLLLLLLPLRGQANTGCYGIPGMPGLPGAPGKDGYDGLPPGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGIPGEPGEEGRYKQKFQSVFT VTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD.

[0112] Thus, the anti-C1q antibodies of the present disclosure can bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of the C1q protein. In some embodiments, the anti-C1q antibodies of the present disclosure bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of human C1q or a homolog thereof, such as mouse, rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pig C1q. In some embodiments, the anti-C1q antibody is a human antibody, a humanized antibody, a chimeric antibody, or a fragment or derivative thereof. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment, such as a Fab fragment.

[0113] All sequences mentioned in the following twenty paragraphs are incorporated by reference into US Patent No. 9,708,394, the antibodies and related compositions disclosed therein are incorporated herein by reference.

[0114] The light chain and heavy chain variable domain sequences of antibody M1 (Mab1) are obtained from U.S. Pat. No. 9,708,394 cited as Incorporated

[0115] Using standard techniques, the nucleic acid and amino acid sequences encoding the light chain variable domain and heavy chain variable domain of antibody M1 were determined. The amino acid sequence of the light chain variable domain of antibody M1 is:

[0116]

[0117] The hypervariable region (HVR) of the light chain variable domain is marked in bold and underlined text. In some embodiments, HVR-L1 of the M1 light chain variable domain has the sequence RASKSINKYLA (SEQ ID NO: 5), HVR-L2 of the M1 light chain variable domain has the sequence SGSTLQS (SEQ ID NO: 6), and HVR-L3 of the M1 light chain variable domain has the sequence QQHNEYPLT (SEQ ID NO: 7).

[0118] The amino acid sequence of the heavy chain variable domain of antibody M1 is:

[0119]

[0120] The hypervariable region (HVR) of the heavy chain variable domain is marked in bold and underlined text. In some embodiments, the HVR-H1 of the M1 heavy chain variable domain has the sequence GYHFTSYWMH (SEQ ID NO: 9), the HVR-H2 of the M1 heavy chain variable domain has the sequence VIHPNSGSINYNEKFES (SEQ ID NO: 10), and the HVR-H3 of the M1 heavy chain variable domain has the sequence ERDSTEVLPMDY (SEQ ID NO: 11).

[0121] The nucleic acid sequence encoding the light chain variable domain was determined to be:

[0122] GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAACAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTG CAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAACATAATGAATACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:12).

[0123] The nucleic acid sequence encoding the heavy chain variable domain was determined to be:

[0124] CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGTCTTCTGGCTACCATTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGTGATTCATCCTAATAGTGGTAGTATTAACTACAAT GAGAAGTTCGAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCGGCGGTCTATTATTGTGCAGGAGAGAGAGATTCTACGGAGGTTCTCCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ IDNO:13).

[0125] Mab1-Fab is the Fab of the Mab1 (M1) antibody.

[0126] Material preservation

[0127] The following materials have been deposited under the Budapest Treaty with the American Type Culture Collection, ATCC Patent Depository, 10801 University Blvd., Manassas, Va. 20110-2209, USA (ATCC):

[0128]

[0129] The hybridoma cell line producing the M1 antibody (Mouse Hybridoma C1qM1 7788-1(M)051613) has been deposited with the ATCC under conditions that ensure availability of culture during the pendency of a patent application for a period of 30 years, or 5 years after the most recent request, or the effective life of the patent, whichever is longer. If the deposit becomes unavailable during that period, the deposit will be replaced. The deposit is available as required by foreign patent laws in countries in which the subject application's counterpart or progeny is filed. It should be understood, however, that the availability of the deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

[0130] Humanized anti-complement C1q antibody (incorporated by reference from U.S. Pat. No. 10,316,081

[0131] The humanized antibodies disclosed herein specifically bind to complement factor C1q and / or C1q protein in the C1 complex of the classical complement pathway. Humanized anti-C1q antibodies may specifically bind to human C1q, human and mouse C1q, rat C1q, or human C1q, mouse C1q and rat C1q.

[0132] In some embodiments, the human heavy chain constant region is a human IgG4 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 26, or having at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% homology to SEQ ID NO: 26. According to Kabat numbering, the human IgG4 heavy chain constant region may comprise an Fc region having one or more modifications and / or amino acid substitutions. In this case, the Fc region comprises an amino acid substitution of leucine to glutamic acid at position 248, wherein such substitution inhibits the interaction of the Fc region with an Fc receptor. In some embodiments, the Fc region comprises an amino acid substitution of serine to proline at position 241, wherein such substitution prevents arm switching in the antibody.

[0133] The amino acid sequence of the heavy chain constant domain of human IgG4 (S241P L248E; corresponding to S108P and L115E in SEQ ID NO: 26) is:

[0134] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:26).

[0135] The antibody may comprise a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 31-34, or an amino acid sequence having at least about 90% homology to an amino acid sequence selected from any one of SEQ ID NOs: 31-34. In certain such embodiments, the light chain variable domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 35-38, or an amino acid sequence having at least about 90% homology to an amino acid sequence selected from any one of SEQ ID NOs: 35-38.

[0136] The amino acid sequence of heavy chain variable domain variant 1 (VH1) is:

[0137] The hypervariable regions (HVRs) of VH1 are indicated in bold and underlined text.

[0138] The amino acid sequence of heavy chain variable domain variant 2 (VH2) is:

[0139] The hypervariable region (HVR) of VH2 is indicated in bold and underlined text.

[0140] The amino acid sequence of heavy chain variable domain variant 3 (VH3) is:

[0141] The hypervariable region (HVR) of VH3 is indicated in bold and underlined text.

[0142] The amino acid sequence of heavy chain variable domain variant 4 (VH4) is:

[0143] The hypervariable region (HVR) of VH4 is indicated in bold and underlined text.

[0144] The amino acid sequence of kappa light chain variable domain variant 1 (Vκ1) is:

[0145] The hypervariable region (HV R) of Vκ1 is indicated in bold and underlined text.

[0146] The amino acid sequence of kappa light chain variable domain variant 2 (Vκ2) is:

[0147] Marked with bold and underlined text.

[0148] The amino acid sequence of kappa light chain variable domain variant 3 (Vκ3) is:

[0149]

[0150] Marked with bold and underlined text.

[0151] The amino acid sequence of kappa light chain variable domain variant 4 (Vκ4) is:

[0152] The hypervariable region (HVR) of Vκ4 is indicated in bold and underlined text.

[0153] The antibody may comprise a light chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NOs: 35-38, while retaining HVR-L1 RASKSINKYLA (SEQ ID NO: 5), HVR-L2 SGSTLQS (SEQ ID NO: 6) and HVR-L3 QQ HNEYPLT (SEQ ID NO: 7). The antibody may comprise a heavy chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NOs: 31-34, while retaining HV R-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYN EKFES (SEQ ID NO: 10) and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0154] In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 35 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 31. In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 36 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 32. In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 37 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody comprises a light chain variable domain amino acid sequence of SEQ ID NO: 38 and a heavy chain variable domain amino acid sequence of SEQ ID NO: 34.

[0155] The full-length antibody Mab2 comprises heavy chain variable domain variant 3 (VH3) (SEQ ID NO: 33) and kappa light chain variable domain variant 3 (Vκ3) (SEQ Id NO: 37). Mab2-Fab is the Fab of the Mab2 antibody.

[0156] The antibody may comprise a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 14; and the light chain comprises the amino acid sequence of SEQ ID NO: 40.

[0157] The amino acid sequence of the heavy chain is:

[0158]

[0159] The hypervariable region (HVR) of VH3 is indicated in bold and underlined text.

[0160] The amino acid sequence of the light chain is:

[0161]

[0162] The complementarity determining regions (CDRs) of SEQ ID NO: 40 are indicated in bold and underlined text.

[0163] In some embodiments, the humanized anti-C1q antibodies of the present disclosure include a heavy chain variable region containing a Fab region and a heavy chain constant region containing an Fc region, wherein the Fab region specifically binds to the C1q protein of the present disclosure, but the Fc region cannot bind to the C1q protein. In some embodiments, the Fc region is from a human IgG1, IgG2, IgG3 or IgG4 isotype. In some embodiments, the Fc region cannot induce complement activity and / or cannot induce antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the Fc region comprises one or more modifications, including but not limited to amino acid substitutions. In certain embodiments, the Fc region of the humanized anti-C1q antibodies of the present disclosure comprises an amino acid substitution at position 248 according to the Kabat numbering convention or a position corresponding to position 248 according to the Kabat numbering convention and / or at position 241 according to the Kabat numbering convention or a position corresponding to position 241 according to the Kabat numbering convention. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 inhibits the interaction of the Fc region with an Fc receptor. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 is an amino acid substitution of leucine to glutamic acid. In some embodiments, the amino acid substitution at position 241 or a position corresponding to position 241 prevents arm switching in the antibody. In some embodiments, the amino acid substitution at position 241 or a position corresponding to position 241 is an amino acid substitution of serine to proline. In certain embodiments, the Fc region of a humanized anti-C1q antibody of the present disclosure comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homology to the amino acid sequence of SEQ ID NO: 47.

[0164] Anti-C1q Fab fragment (e.g., FabA)

[0165] All anti-Clq antibody Fab fragment sequences are incorporated by reference from US Patent No. 10,723,788, the antibodies and related compositions disclosed in which are hereby incorporated by reference.

[0166] In certain embodiments, the present disclosure provides binding sites comprising a heavy chain (V H / C H 1) and light chain (V L / C L ), wherein the anti-C1q antibody Fab fragment has six complementary determining regions (CDRs), V L and V H Three each (HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3). The heavy chain of the antibody Fab fragment is truncated after the first heavy chain domain of IgG1 (SEQ ID NO: 39) and comprises the following amino acid sequence:

[0167]

[0168]

[0169] The complementarity determining regions (CDRs) of SEQ ID NO: 39 are indicated in bold and underlined text.

[0170] The light chain domain of the antibody Fab fragment comprises the following amino acid sequence (SEQ ID NO: 40):

[0171]

[0172] The complementarity determining regions (CDRs) of SEQ ID NO: 40 are indicated in bold and underlined text.

[0173] FabA is an anti-C1q antibody Fab fragment comprising a heavy chain domain comprising SEQ ID NO:39 and a light chain domain comprising SEQ ID NO:40.

[0174] Mab1-Fab is the Fab of the Mab1 (M1) antibody.

[0175] Mab2-Fab is the Fab of the Mab2 antibody.

[0176] Anti-C1q one-armed antibody

[0177] All anti-Clq one-armed antibody sequences are disclosed in US Patent Application No. 63 / 288,883 and US Patent Application No. 63 / 288,885, the antibodies and related compositions disclosed therein are hereby incorporated by reference.

[0178] In certain aspects, the present disclosure provides antibodies that bind to proteins in the complement cascade, such as C1q proteins. The antibody that binds to C1q comprises a single C1q antigen binding arm and an Fc region. The single C1q antigen binding arm may comprise a light chain variable domain and a heavy chain variable domain. The Fc region may comprise a complex of a first Fc polypeptide and a second Fc polypeptide. The Fc region may comprise an Fcγ receptor binding site mutation. The antibody may belong to the IgG4 category. In some embodiments, one but not both of the Fc polypeptides is an N-terminally truncated heavy chain. In some embodiments, the Fcγ receptor is FcγRI, FcγRII or FcγRIII, preferably FcγRI. The Fcγ receptor binding site mutation may comprise an IgG4 L115E mutation.

[0179]

[0180] The complementarity determining regions (CDRs) of SEQ ID NO: 40 are marked in bold and underlined text. In some embodiments, HVR-L1 of the light chain variable domain has the sequence RASK SINKYLA (SEQ ID NO: 5), HVR-L2 of the light chain variable domain has the sequence SGSTLQS (SEQ ID NO: 6), and HVR-L3 of the light chain variable domain has the sequence QQHNEYPLT (SEQ ID NO: 7).

[0181] The light chain of the one-armed antibody may comprise the following light chain variable domain amino acid sequence:

[0182]

[0183] The one-armed antibody may comprise a light chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 10, preferably while retaining HVR-L1 RA SKSINKYLA (SEQ ID NO: 5), HVR-L2 SGSTLQS (SEQ ID NO: 6) and HVR-L3 QQHNEYPLT (SEQ ID NO: 7).

[0184] The one-armed antibody may comprise the following amino acid sequence of kappa light chain variable domain variant 1 (Vκ1):

[0185] The hypervariable region (HV R) of Vκ1 is indicated in bold and underlined text.

[0186] The one-armed antibody may comprise the following amino acid sequence of kappa light chain variable domain variant 2 (Vκ2):

[0187] The hypervariable region (HVR) of Vκ2 is indicated in bold and underlined text.

[0188] The one-armed antibody may comprise the following amino acid sequence of kappa light chain variable domain variant 3 (Vκ3):

[0189] The hypervariable region (HVR) of Vκ3 is indicated in bold and underlined text.

[0190] The one-armed antibody may comprise the following amino acid sequence of kappa light chain variable domain variant 4 (Vκ4):

[0191] The hypervariable region (HVR) of Vκ4 is indicated in bold and underlined text.

[0192] The one-armed antibody may comprise a light chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NOs: 11-14, while retaining HVR-L1 RASKS INKYLA (SEQ ID NO: 5), HVR-L2 SGSTLQS (SEQ ID NO: 6) and HVR-L3 QQHNEYPLT (SEQ ID NO: 7).

[0193] The antibody may belong to the IgG4 class. The sequence of the IgG4 heavy chain is

[0194]

[0195] The domains of IgG4 are as follows: CH1: 1-98, hinge: 99-110, CH2: 111-220 and CH3: 221-327. IgG4 may comprise mutations. For example, S108P mutation (for IgG4 arm exchange), L115E mutation (for FcR binding), T246W mutation (for knob-into-hole mutation), T246S mutation (for knob-into-hole mutation), L248A mutation (for knob-into-hole mutation), Y187V mutation (for knob-into-hole mutation), and / or N187A (deglycosylated for FcR binding), N187Q (deglycosylated for FcR binding) or N187G (deglycosylated for FcR binding).

[0196] One heavy chain of the one-armed antibody (heavy chain 1 domain) of the one-armed antibody may comprise the following amino acid sequence (SEQ ID NO: 2):

[0197]

[0198] The complementarity determining regions (CDRs) of SEQ ID NO:2 are indicated in bold and underlined text. The knob-in-hole T366W mutation (corresponding to the IgG4 T246W mutation) in SEQ ID NO:2 is indicated in underlined text. The S241P (for IgG4 arm switch, corresponding to S108P) and L248E (for FCR, corresponding to the L115E mutation) mutations are indicated in bold text. In some embodiments, the HVR-H1 of the heavy chain variable domain has the sequence GYHFTSYWMH (SEQ ID NO:9), the HVR-H2 of the heavy chain variable domain has the sequence VIHPNSGS INYNEKFES (SEQ ID NO:10), and the HVR-H3 of the heavy chain variable domain has the sequence ERDSTEVLPMDY (SEQ ID NO:11).

[0199] One heavy chain of the one-armed antibody (heavy chain 1 domain) of the one-armed antibody may comprise the following amino acid sequence (SEQ ID NO: 20):

[0200]

[0201] The complementarity determining regions (CDRs) of SEQ ID NO: 20 are marked in bold and underlined text. The knob-in-hole T366W mutation (corresponding to the IgG4 T246W mutation) in SEQ ID NO: 20 is marked in underlined text. The S241P (corresponding to S108P for IgG4 arm swap) mutation and L248 (corresponding to the L115E mutation) are marked in bold text.

[0202] The antibody may belong to the IgG1 class. The sequence of the IgG1 heavy chain is

[0203]

[0204] The domains of IgG1 are as follows: CH1: 1-98, hinge: 99-110, CH2: 111-223 and CH3: 224-330. IgG1 may include mutations. For example, L117A mutation (for FcR binding), L118A mutation (for FcR binding), T249W mutation (for knob-into-hole mutation), T249S mutation (for knob-into-hole mutation), L251A mutation (for knob-into-hole mutation) and / or Y290V mutation (for knob-into-hole mutation).

[0205] One heavy chain of the one-armed antibody (heavy chain 1 domain) of the one-armed antibody may comprise the following amino acid sequence (SEQ ID NO: 21):

[0206]

[0207] The complementarity determining regions (CDRs) of SEQ ID NO:21 are marked in bold and underlined text. The knob-in-hole T366W mutation (corresponding to the IgG1 T249W mutation) in SEQ ID NO:21 is marked in underlined text. The L234A (corresponding to the IgG1 L117A mutation) and L235A (corresponding to the IgG1 L1178 mutation) mutations are marked in bold text.

[0208] The heavy chain 1 of the one-armed antibody may comprise the following heavy chain variable domain amino acid sequence:

[0209]

[0210] The one-armed antibody may comprise a heavy chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 15, preferably while retaining HVR-H1GYHFTSYWMH (SEQ ID NO: 9), HVR-H2VIHPNSGSINYNEKFES (SEQ ID NO: 10) and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0211] The one-armed antibody may comprise the following amino acid sequence of heavy chain variable domain variant 1 (VH1):

[0212] The hypervariable regions (HVRs) of VH1 are indicated in bold and underlined text.

[0213] The one-armed antibody may comprise the following amino acid sequence of heavy chain variable domain variant 2 (VH2): The hypervariable region (HVR) of VH2 is indicated in bold and underlined text.

[0214] The one-armed antibody may comprise the following amino acid sequence of heavy chain variable domain variant 3 (VH3):

[0215] The hypervariable region (HVR) of VH3 is indicated in bold and underlined text.

[0216] The one-armed antibody may comprise the following amino acid sequence of heavy chain variable domain variant 4 (VH4): The hypervariable region (HVR) of VH4 is indicated in bold and underlined text.

[0217] The one-armed antibody may comprise a heavy chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NOs: 16-19, while retaining HVR-H1 GYHF TSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO: 10) and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0218] The second heavy chain of the one-armed antibody (heavy chain 2 domain) of the one-armed antibody is N-terminally truncated and may comprise the following amino acid sequence (SEQ ID NO: 3):

[0219]

[0220] There is no heavy chain variable domain and no CDRs in SEQ ID NO: 3. The knob-in-hole T366S / L368A / Y407V mutations in SEQ ID NO: 3 are marked in underlined text. The S241P and L248E mutations are marked in bold text.

[0221] The second heavy chain of the one-armed antibody (heavy chain 2 domain) of the one-armed antibody is N-terminally truncated and may comprise the following amino acid sequence (SEQ ID NO: 42):

[0222]

[0223]

[0224] The second heavy chain of the one-armed antibody (heavy chain 2 domain) of the one-armed antibody is N-terminally truncated and may comprise the following amino acid sequence (SEQ ID NO: 43):

[0225]

[0226] The heavy chain variable domain and CDRs are absent in SEQ ID NO: 43. The knob-in-hole T366S / L368A / Y407V mutations in SEQ ID NO: 43 are indicated in underlined text. The L234A and L235A mutations are indicated in bold text.

[0227] The second heavy chain (heavy chain 2 domain) of the antibody may comprise any one of the following amino acid sequences (SEQ ID NOs: 44-47):

[0228]

[0229]

[0230]

[0231] The second heavy chain (heavy chain 2 domain) of the antibody may comprise any one of the following amino acid sequences (SEQ ID NOs: 48-51):

[0232]

[0233]

[0234] The CDRs in the second heavy chain variable domain were mutated to prevent binding to CIq in SEQ ID NOs:44-51. The CDR mutations are indicated in bold and underlined text. The knob-in-hole T366S / L368A / Y407V mutations in SEQ ID NOs:44-51 are indicated in underlined text.

[0235] In some embodiments, the antibody that binds to CIq comprises:

[0236] a light chain domain, the light chain domain comprising the amino acid sequence of SEQ ID NO: 40;

[0237] A first heavy chain domain, the first heavy chain domain comprising the amino acid sequence of SEQ ID NO: 2; and

[0238] The second heavy chain domain comprises the amino acid sequence of SEQ ID NO: 3; the second heavy chain domain is an N-terminally truncated heavy chain.

[0239] Nucleic acids, vectors and host cells

[0240] Antibodies suitable for use in the methods of the present disclosure can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In some embodiments, isolated nucleic acids having nucleotide sequences encoding any of the antibodies of the present disclosure are provided. Such nucleic acids may encode a V L / C L The amino acid sequence of the anti-C1q antibody and / or V H / C H 1. In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. Host cells containing such nucleic acids can also be provided. The host cells can contain the following (e.g., have been transduced with the following): (1) a V vector encoding an antibody; L / C L The amino acid sequence and V containing antibodyH / C H 1, or (2) a vector containing a nucleic acid encoding an antibody V L / C L A first vector containing a nucleic acid encoding an amino acid sequence of H / C H In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In some embodiments, the host cell is a bacterium, such as Escherichia coli.

[0241] Disclosed herein are methods for preparing anti-C1q antibodies. The methods include culturing a host cell of the disclosure containing a nucleic acid encoding an anti-C1q antibody under conditions suitable for antibody expression. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).

[0242] To recombinantly produce the humanized anti-C1q antibodies of the present disclosure, nucleic acids encoding the antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibodies).

[0243] Suitable vectors containing nucleic acid sequences encoding any antibody or fragment polypeptides thereof (including antibodies) disclosed herein include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although the cloning vector selected can vary according to the host cell to be used, useful cloning vectors generally have the ability to self-replicate, can have a single target for a specific restriction endonuclease, and / or can carry genes for markers that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and derivatives thereof, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors can be obtained from commercial suppliers, such as BioRad, Stratagene, and Invitrogen.

[0244] Vectors containing nucleic acids of interest can be introduced into host cells by any of a number of suitable methods, including electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE dextran or other substances; microprojectile bombardment; lipid infection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of the vector or polynucleotide to introduce will generally depend on the characteristics of the host cell. In some embodiments, the vector comprises a nucleic acid comprising one or more amino acid sequences encoding an anti-C1q antibody of the present disclosure.

[0245] Host cells suitable for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells. For example, the anti-C1q antibodies of the present disclosure can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expressing antibody fragments and polypeptides in bacteria (e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; and Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describes the expression of antibody fragments in E. coli). In other embodiments, the antibodies of the present disclosure can be produced in eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NS0, Sp20 cells) (e.g., U.S. Patent Application No. 14 / 269,950; U.S. Patent No. 8,981,071; Eur J Biochem. 1991 Jan 1; 195(1):235-42). After expression, the antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0246] Pharmaceutical compositions, formulations and administration

[0247] The present disclosure as a whole relates to pharmaceutical compositions comprising the anti-C1q antibodies (including antibody Fab fragments and antibody derivatives) disclosed herein. As used herein, the expression "pharmaceutical composition (also referred to as "pharmaceutical preparation") means a combination of at least one active ingredient (e.g., an anti-C1q antibody disclosed herein, including antibody Fab fragments and antibody derivatives) and at least one inactive ingredient, which inactive ingredient is suitable for therapeutic administration to humans or non-human animals when combined with the active ingredient and / or one or more additional inactive ingredients.

[0248] Formulations of anti-C1q antibodies (including antibody Fab fragments and antibody derivatives) are identified. Such formulations are isotonic in nature and are formulated at high concentrations (e.g., greater than about 75 mg / ml). In certain embodiments, the formulations exhibit a high level of stability. Such formulations are also capable of meeting the subvisible particulate matter USP for ophthalmic products. <789> An example of a selected formulation is at least 75 mg / ml (e.g., 100 mg / mL, 125 mg / mL, 150 mg / mL, 175 mg / mL, 200 mg / mL, 225 mg / mL, 250 mg / mL anti-C1q antibody (or antibody Fab fragment and antibody derivative)) formulated in 5 mM to 25 mM sodium citrate or sodium succinate (e.g., 5 mM, 10 mM, 15 The pharmaceutical composition may also include 30mM to 50mM NaCl (e.g., 30mM, 40mM, 50mM NaCl).

[0249] The pharmaceutical composition may include a sodium citrate or sodium succinate buffer. The sodium citrate or sodium succinate may be about 5 mM to about 25 mM. In some embodiments, the sodium citrate or sodium succinate may be about 5 mM to about 20 mM. In some embodiments, the sodium citrate or sodium succinate may be about 5 mM to about 10 mM. In some embodiments, the sodium citrate may be about 10 mM. In some embodiments, the sodium succinate may be about 10 mM.

[0250] The pharmaceutical composition can also include a stabilizer that reduces the osmotic pressure molar concentration and keeps the composition isotonic, such as trehalose. Trehalose can be between about 3% to about 7% (w / v). In some embodiments, trehalose can be 3%, 4%, 5%, 6% or 7% (w / v). In some embodiments, trehalose can be 5% (w / v).

[0251] The pharmaceutical composition may also include a surfactant such as PS20 or PS80 to minimize aggregation (subvisible particle reduction). PS20 or PS80 may be 0.04%-0.06%. In some embodiments, PS20 may be 0.06% (w / v). In some embodiments, PS80 may be 0.06% (w / v).

[0252] The pH of the pharmaceutical composition may be 6.4-7.5. In some embodiments, the pharmaceutical composition may have a pH of about 7.2.

[0253] Optionally, the pharmaceutical composition may further comprise 30 mM to 50 mM NaCl. In some embodiments, the pharmaceutical composition may optionally comprise 40 mM NaCl.

[0254] In certain aspects, provided herein is a pharmaceutical composition comprising: (a) at least 75 mg / ml of an anti-C1q antibody; 5 mM to 25 mM sodium citrate or sodium succinate; (c) 3% to 7% trehalose; and (d) 0.04% to 0.06% polysorbate 20 or polysorbate 80, wherein the pH of the pharmaceutical composition is 6.4-7.5.

[0255] In certain embodiments, the pharmaceutical composition comprises at least 75 mg / ml, at least 100 mg / ml, at least 125 mg / ml, at least 150 mg / ml, at least 175 mg / ml, at least 200 mg / ml, at least 225 mg / ml, or at least 250 mg / ml of anti-C1q antibodies, including antibody Fab fragments and antibody derivatives. The pharmaceutical composition may comprise 75 mg / ml to 300 mg / ml, 100 mg / ml to 300 mg / ml, or 125 mg / ml to 250 mg / ml of anti-C1q antibodies, including antibody Fab fragments and antibody derivatives. In some embodiments, the pharmaceutical composition may comprise about 75 mg / ml, 100 mg / ml, 125 mg / ml, about 150 mg / ml, about 175 mg / ml, about 200 mg / ml, 225 mg / ml, or about 250 mg / ml of anti-C1q antibody Fab fragments. The pharmaceutical composition may comprise 100 mg / ml to 125 mg / ml, 125 mg / ml to 150 mg / ml, 150 mg / ml to 175 mg / ml, 175 mg / ml to 200 mg / ml or 200 mg / ml to 250 mg / ml of anti-C1q antibody, including antibody Fab fragments and antibody derivatives.

[0256] In certain embodiments, the pharmaceutical composition comprises about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM or about 25mM sodium citrate or sodium succinate. Sodium citrate can be 10mM. Sodium succinate can be 5mM to 20mM. The pharmaceutical composition can comprise 5mM to 10mM, 10mM to 15mM, 15mM to 20mM or 20mM to 25mM sodium citrate or sodium succinate.

[0257] In certain embodiments, the pharmaceutical composition comprises about 3%, 4%, 5%, 6% or 7% stabilizer. The pharmaceutical composition may comprise 3%-4%, 4%-5%, 5%-6% or 6%-7% trehalose. Stabilizers such as trehalose increase osmotic pressure molarity. Trehalose may be present in an amount of about 4% to about 6%, preferably about 5% (w / v).

[0258] In certain embodiments, the pharmaceutical composition comprises about 0.04%, 0.05% or 0.06% surfactant to minimize aggregation (subvisible particle reduction). The surfactant can be polysorbate 20 (PS20) or polysorbate 80 (PS80). The pharmaceutical composition can comprise 0.04% to 0.05% or 0.05% to 0.06% polysorbate 20 or polysorbate 80.

[0259] In certain embodiments, the pH of the pharmaceutical composition is about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. The pH of the pharmaceutical composition may be 6.0-6.5 or 6.5-7.0 or 7.0-7.5.

[0260] Optionally, the pharmaceutical composition also comprises 30mM to 50mM NaCl. The pharmaceutical composition may comprise about 30mM, about 35mM, about 40mM, about 45mM or about 50mM NaCl. For isotonicity, NaCl may be added. The pharmaceutical composition may comprise 30mM to 35mM, 35mM to 40mM, 40mM to 45mM or 45mM to 50mM NaCl. The amount of NaCl may depend on the amount of trehalose in the formulation. The more trehalose is added, the less NaCl is required. The amount of NaCl may also depend on the antibody concentration.

[0261] In certain embodiments, the viscosity of the pharmaceutical composition does not exceed 10 cP at 25° C. The viscosity of the pharmaceutical composition may be about 1 cP, about 2 cP, about 3 cP, about 4 cP, about 5 cP, about 6 cP, about 7 cP, about 8 cP, about 9 cP, or about 10 cP at 25° C. The viscosity of the pharmaceutical composition may be 1 cp to 5 cp or 5 cp to 10 cp at 25° C.

[0262] The pharmaceutical composition can be stable for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 20 weeks, at least 21 weeks, at least 22 weeks, at least 23 weeks, or at least 24 weeks. The pharmaceutical composition can be stable at a temperature equal to or greater than 25°C. In some embodiments, the pharmaceutical composition can be stable at a temperature between about 25°C and about 40°C. The pharmaceutical composition can be stable at a temperature of about 25°C, about 30°C, about 35°C, or about 40°C.

[0263] In some embodiments, the pharmaceutical composition is stable for at least 1 year, at least 1.5 years, or at least two years. The pharmaceutical composition may be stable at a temperature of -20°C to 25°C. The pharmaceutical composition may be stable at a temperature of about -20°C, about -15°C, about -10°C, about -5°C, about 0°C, about 2°C, about 4°C, about 5°C, about 8°C, about 10°C, about 15°C, about 20°C, or about 25°C. The pharmaceutical composition may be stable at a temperature of 2°C to 8°C. The pharmaceutical composition may be stable at a temperature greater than or equal to 25°C, such as between about 25°C to about 40°C, preferably about 25°C, about 30°C, about 35°C, or about 40°C.

[0264] The pharmaceutical composition can be stable after at least one, at least two, at least three, at least four, or at least five freeze / thaw cycles. The pharmaceutical composition can be stable after continuous stirring for at least one, at least two, or at least three days.

[0265] In certain embodiments, the pharmaceutical composition is prepared for intravitreal or intraocular injection. The pharmaceutical composition may be contained in a syringe. In some embodiments, the pharmaceutical composition is pre-filled in a syringe. In some embodiments, the syringe has a fill volume of no more than 300 microliters. In some embodiments, the syringe delivers a volume of 25 microliters-100 microliters.

[0266] In certain embodiments, the pharmaceutical composition is prepared for subcutaneous injection. The pharmaceutical composition can be contained in a syringe, an automatic injector, or a pen. In some embodiments, the syringe, automatic injector, or pen has a filling volume of no more than 10 ml.

[0267] In certain embodiments, the pharmaceutical composition is prepared for intravenous injection.The pharmaceutical composition can be contained in an intravenous solution bag.

[0268] In certain embodiments, the pharmaceutical compositions provided herein show a high level of stability. As used herein with respect to pharmaceutical compositions, the term "stable" means that the antibody in the pharmaceutical composition still maintains an acceptable degree of structure and / or function and / or biological activity after storing a specified amount of time. Even if the antibody included in the composition cannot maintain 100% of its structure and / or function and / or biological activity after storing a specified amount of time, the composition can also be stable. In some cases, after storing a specified amount of time, the structure and / or function and / or biological activity of the antibody maintains about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% and can be considered as "stable".

[0269] Stability can be measured in particular by measuring the percentage of natural antibodies retained in the composition after storing a specified amount of time at a given temperature. "Native antibody" refers to an antibody with a "natural conformation", which refers to the secondary, tertiary and / or quaternary structure of the antibody in its biologically active state. For example, natural antibodies are in monomeric form. Several methods based on physical parameters can be used to analyze and quantify aggregated antibodies and to measure the amount of aggregated antibodies and monomeric antibodies. The percentage of natural or monomeric antibodies can be measured in particular by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SE-HPLC]). The phrase "acceptable degree of stability" used herein means that after storing a specified amount of time at a given temperature, at least 90% of the natural or monomeric form of antibodies can be detected in the composition. In certain embodiments, after storing a specified amount of time at a given temperature, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the natural or monomeric form of antibodies can be detected in the composition. The specified amount of time after stability is measured can be at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. The pharmaceutical composition can be stable for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 20 weeks, at least 21 weeks, at least 22 weeks, at least 23 weeks, or at least 24 weeks. The pharmaceutical composition can be stable for a minimum of two years. When evaluating stability, the temperature at which the pharmaceutical composition can be stored can be any temperature from about -20°C to 25°C, for example, at about -20°C, about -15°C, about -10°C, about -5°C, about 0°C, about 2°C, about 4°C, about 5°C, about 8°C, about 10°C, about 15°C, about 20°C, or about 25°C. The pharmaceutical composition can be stable at a temperature of 2°C to 8°C. The pharmaceutical composition can be stable at a temperature greater than or equal to 25°C, such as between about 25°C to about 40°C, preferably about 25°C, about 30°C, about 35°C, or about 40°C. For example, if greater than about 90%, 95%, 96%, or 97% of the natural or monomeric antibodies are detected by SE-HPLC after storage at 5°C for 3 months, the pharmaceutical composition can be considered stable. The pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96% or 97% of native or monomeric antibody is detected by SE-HPLC after storage at 5°C for 6 months.The pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the native or monomeric antibodies are detected by SE-HPLC after 9 months storage at 5°C. The pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the native or monomeric antibodies are detected by SE-HPLC after 3 months storage at 25°C. The pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the native or monomeric antibodies are detected by SE-HPLC after 6 months storage at 25°C. The pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the native or monomeric antibodies are detected by SE-HPLC after 9 months storage at 25°C.

[0270] Other methods can be used to assess the stability of the pharmaceutical compositions disclosed herein, such as SEC-HPLC, visual particle inspection, and light shielding by HIAC, for measuring aggregation, visible particles, and sub-visible particles, respectively. Another stability indicator method is cation exchange HPLC, which is also used to detect changes in charge variant distribution for stability assessment.

[0271] Stability can also be assessed by measuring the biological activity and / or binding affinity of the antibody to its target. For example, if after storage at, e.g., 5°C, 25°C, 40°C, etc. for a specified amount of time (e.g., 1 to 12 months), the anti-C1q antibody contained in the composition binds to C1q with an affinity that is at least 50%, 60%, 70%, 80%, 90%, 95% or more of the binding affinity of the anti-C1q antibody contained in the composition prior to said storage, then the compositions disclosed herein can be considered stable. Additional methods for assessing the stability of antibodies in the compositions are described in the Examples presented below.

[0272] The stability of a pharmaceutical composition can be assessed based on the evolution of its physical and chemical parameters over time. Physical stability includes visual and olfactory data as well as tracking of invisible particles. To study chemical stability, analytical methods are used to achieve separation and even quantification of any degradation products. Monitoring of the concentration of the active ingredient and the appearance of degradation products over time is accomplished using methods known to those of ordinary skill in the art, including, for example, by UV280 (for concentration) and SEC-HPLC, sodium dodecyl sulfate capillary gel electrophoresis (CE-SDS) and cation exchange HPLC (for the appearance of degradation products). If USP is met, <789> 、USP <787> or USP <788> limit, the pharmaceutical composition may be stable.

[0273] As used herein, with respect to the pharmaceutical compositions disclosed herein, a low level of viscosity will exhibit an absolute viscosity of less than about 20 centipoise (cP) at 25° C. For example, if the composition exhibits an absolute viscosity of about 19 cP, about 18 cP, about 17 cP, about 16 cP, about 15 cP, about 14 cP, about 13 cP, about 12 cP, about 11 cP, about 10 cP, about 9 cP, about 8 cP, about 7 cP, about 6 cP, about 5 cP, about 4 cP or less at 25° C., the pharmaceutical compositions disclosed herein will be considered to have "low viscosity". As used herein, with respect to the pharmaceutical compositions disclosed herein, a medium level of viscosity will exhibit an absolute viscosity between about 30 cP and about 20 cP at 25° C. For example, a pharmaceutical composition disclosed herein would be considered to have "medium viscosity" if the composition exhibits an absolute viscosity of about 30 cP, about 29 cP, about 28 cP, about 27 cP, about 26 cP, about 25 cP, about 24 cP, about 23 cP, about 22 cP, about 21 cP, or about 20 cP at 25°C when measured using standard viscosity measurement techniques.

[0274] In some embodiments, the osmotic pressure molar concentration of the pharmaceutical composition is within the physiological osmotic pressure molar concentration range of 250mOsm / kg-400mOsm / kg. In some embodiments, the viscosity of the pharmaceutical composition does not exceed 30cP at 25°C. In some embodiments, the viscosity of the pharmaceutical composition does not exceed 15cP at 25°C. In some embodiments, the viscosity of the pharmaceutical composition is about 10cP, about 11cP, about 12cP, about 13cP, about 14cP, about 15cP, about 16cP, about 17cP, about 18cP, about 19cP, about 20cP, about 21cP, about 22cP, about 23cP, about 24cP, about 25cP, about 26cP, about 27cP, about 28cP, about 29cP or about 30cP at 25°C.

[0275] Application

[0276] Disclosed herein are methods of administering a pharmaceutical composition comprising at least 75 mg / ml of an anti-C1q antibody. The pharmaceutical composition may also comprise 5 mM to 25 mM sodium citrate or sodium succinate; 3% to 7% trehalose; and / or 0.04% to 0.06% polysorbate 20 or polysorbate 80. The pH of the pharmaceutical composition may be 6.4-7.5. The anti-C1q antibody may comprise a light chain variable domain and a heavy chain variable domain. The antibody may bind at least human C1q, mouse C1q, or rat C1q. The antibody may be a humanized antibody, a chimeric antibody, or a human antibody. The antibody may be a monoclonal antibody, an antibody fragment thereof, and / or an antibody derivative thereof. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment, such as a Fab fragment. The light chain variable domain of the antibody, its antibody fragment and / or its antibody derivative comprises HVR-L1, HVR-L2 and HVR-L3 of the monoclonal antibody M1 produced by the hybridoma cell line deposited with Accession No. PTA-120399. The heavy chain variable region of the antibody, its antibody fragment and / or its antibody derivative comprises HVR-H1, HVR-H2 and HVR-H3 of the monoclonal antibody M1 produced by the hybridoma cell line deposited with ATCC Accession No. PTA-120399.

[0277] In some embodiments, the amino acid sequences of the light chain variable domain and the heavy chain variable domain comprise one or more of SEQ ID NO: 5 of HVR-L1, SEQ ID NO: 6 of HVR-L2, SEQ ID NO: 7 of HVR-L3, SEQ ID NO: 9 of HVR-H1, SEQ ID NO: 10 of HVR-H2, and SEQ ID NO: 11 of HVR-H3.

[0278] The antibody may comprise a light chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 4, preferably while retaining HVR-L1 RASKSINKYLA (SEQ ID NO: 5), HVR-L2 SGSTLQS (SEQ ID NO: 6) and HVR-L3 QQHNEYPLT (SEQ ID NO: 7). The antibody may comprise a heavy chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 8, preferably while retaining HVR-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSIN YNEKFES (SEQ ID NO: 10) and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0279] In certain embodiments of the compositions and methods provided herein, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is administered intravenously. In certain embodiments of the compositions and methods provided herein, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is administered subcutaneously. In certain embodiments, the pharmaceutical composition is formulated for intramuscular administration. In certain embodiments, the pharmaceutical composition is administered intramuscularly. In some embodiments, the pharmaceutical composition is formulated for infraorbital, intravitreal, intraocular, subconjunctival, retrobulbar, peribulbar and / or intrathecal administration. In certain embodiments, the pharmaceutical composition is administered by infraorbital, intravitreal, intraocular, subconjunctival, retrobulbar, peribulbar and / or intrathecal injection.

[0280] In certain embodiments, the pharmaceutical composition is administered in a delivery volume of no more than 3 ml, 2.5 ml, 2 ml, 1.5 ml, or 1 ml. In some embodiments, the pharmaceutical composition is administered in a delivery volume of no more than 2 ml. In some embodiments, the pharmaceutical composition is administered in a delivery volume of no more than 300 microliters. In some embodiments, the pharmaceutical composition is administered in a delivery volume of no more than 25 microliters, 50 microliters, 75 microliters, or 100 microliters.

[0281] In certain aspects, the disclosure relates to a syringe comprising a pharmaceutical composition disclosed herein. The syringe can be used for subcutaneous, intravitreal or intraocular injection. In some embodiments, the syringe has a delivery volume of no more than 10 μl. In some embodiments, the syringe includes a needle of size no greater than 25G (25 gauge). In some embodiments, the syringe is an automatic reusable fixed dose pen. In other embodiments, the syringe is an automatic reusable variable dose pen. In another embodiment, the syringe is an automatic disposable fixed dose automatic syringe. In some embodiments, the syringe has a delivery volume of no more than 300 microliters. The syringe can be a syringe.

[0282] In certain aspects, the present disclosure relates to a pre-filled syringe comprising a pharmaceutical composition disclosed herein.The pre-filled syringe may have a delivery volume of no more than 300 microliters.

[0283] In some embodiments, the pharmaceutical composition provided herein can be contained in any container suitable for storing drugs and other therapeutic compositions. For example, the pharmaceutical composition can be contained in a sealed and sterilized plastic or glass container with a specified volume, such as a vial, an ampoule, a syringe, a cartridge or a bottle. Different types of vials can be used to hold the composition provided herein, including, for example, transparent and opaque (e.g., amber) glass or plastic vials. Similarly, any type of syringe can be used to hold and / or apply the pharmaceutical composition disclosed herein.

[0284] The pharmaceutical compositions provided herein can be contained in plastic polymer syringes that are silicon-free and low in particulate levels, i.e., comply with USP for subvisible particulate matter. <789> Limits (less than fifty particles per ml for 10 μm particles and less than five particles per ml for 25 μm particles) (see USP <789> ). Particulate matter in ophthalmic solutions. USP 35–NF 30. MD, USA: The United States Pharmacopeial Convention; 2012). For example, Terumo's i-coating stopper technology eliminates the need for silicone oil in syringe systems while providing consistent and predictable break-out and glide forces. Eliminating the use of silicone oil in prefilled syringe (PFS) systems significantly reduces subvisible particle loads and allows for meeting stringent particle requirements such as USP <789> Particulate matter in ophthalmic solutions.

[0285] The pharmaceutical compositions provided herein can be contained in a "normal tungsten" syringe or a "low tungsten" syringe. As will be appreciated by those of ordinary skill in the art, the method of preparing a glass syringe generally involves the use of a hot tungsten rod, which is used to pierce the glass, thereby creating a hole from which liquid can be drawn and discharged from the syringe. This method results in trace amounts of tungsten being deposited on the inner surface of the syringe. Subsequent washing and other processing steps can be used to reduce the amount of tungsten in the syringe. As used herein, the term "normal tungsten" means that the syringe contains greater than 500 parts per billion (ppb) of tungsten. The term "low tungsten" means that the syringe contains less than 500 ppb of tungsten. For example, a low tungsten syringe may contain less than about 490 ppb, 480 ppb, 470 ppb, 460 ppb, 450 ppb, 440 ppb, 430 ppb, 420 ppb, 410 ppb, 390 ppb, 350 ppb, 300 ppb, 250 ppb, 200 ppb, 150 ppb, 100 ppb, 90 ppb, 80 ppb, 70 ppb, 60 ppb, 50 ppb, 40 ppb, 30 ppb, 20 ppb, 10 ppb, or less ppb of tungsten.

[0286] The rubber plungers used in syringes and the rubber stoppers used to close the openings of vials can be coated to prevent contamination of the drug contents of the syringes or vials and / or to maintain their stability. Therefore, according to certain embodiments, the pharmaceutical compositions provided herein can be contained in a syringe having a coated plunger, or in a vial sealed with a coated rubber stopper. For example, the plunger or stopper can be coated with a fluorocarbon film. Examples of coated stoppers and / or plungers suitable for vials and syringes containing the pharmaceutical compositions disclosed herein are mentioned in, for example, U.S. Patents Nos. 4,997,423; 5,908,686; 6,286,699; 6,645,635; and 7,226,554, the contents of which are incorporated herein by reference in their entirety. Specific exemplary coated rubber stoppers and plungers that can be used in the methods disclosed herein can be found under the trade name Commercially available from West Pharmaceutical Services, Inc. (Lionville, Pa.).

[0287] In certain embodiments, the pharmaceutical composition may be administered to a patient by intravitreal or intraocular injection.

[0288] In certain embodiments, the pharmaceutical composition can be administered to a patient by a parenteral route such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, suborbital, intravitreal, intraocular, subconjunctival, retrobulbar, peribulbar and / or intrathecal injection). Many reusable pens and / or automatic syringe delivery devices can be used for subcutaneous delivery of the pharmaceutical compositions disclosed herein. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISSETRONIC TM pen (Disetronic Medical Systems, Bergdorf, Sweden), HUMALOG MIX 75 / 25 TM pen、HUMALOG TM pen, HU MALIN 70 / 30 TM pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOP EN JUNIOR TM (NovoNordisk,Copenhagen,Denmark)、BD TMpen(Becton Dickinson,Franklin Lakes,NJ)、OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), to name a few. Examples of disposable pen and / or auto-injector delivery devices for use in subcutaneous delivery of the pharmaceutical compositions disclosed herein include, but are not limited to, SOLOS TAR TM pen (sanofi-aventis), FLEXPEN TM (NovoNordisk) and KWIKP EN TM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, Calif.), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA TM Pen (Abbott Labs, Abbott Park, Ill.), to name a few.

[0289] It is also contemplated herein to use a microinjector to deliver the pharmaceutical compositions disclosed herein. As used herein, the term "microinjector" means a subcutaneous delivery device designed to slowly administer a large volume (e.g., up to about 2.5 mL or more) of a therapeutic composition over an extended period of time (e.g., about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes or more). See, e.g., U.S. Patent No. 6,629,949; No. 6,659,982; and Meehan et al., J. Controlled Release 46: 107-116 (1996).

[0290] Treatment

[0291] In certain aspects, the present disclosure relates to methods for preventing, reducing the risk of developing, or treating a disease or condition associated with a complement pathway, comprising administering a pharmaceutical composition disclosed herein. The present disclosure also generally relates to methods for preventing, reducing the risk of developing, or treating a disease and condition associated with a complement pathway, comprising administering a pharmaceutical composition using a syringe, a syringe, or an intravenous solution bag disclosed herein.

[0292] The methods of the present invention modulate the immune response to the diseases disclosed herein by administering a complement antagonist. For example, without being bound by theory, immature astrocytes induce expression of C1q protein in neurons during development. Inflammatory mediators such as complement factors are usually expressed at very low levels in healthy brain tissue, but can be rapidly induced by various brain insults such as infection, ischemia, and injury. Activation of C1q, C1r, and C1s contributes to the inflammatory response, leading to synaptic loss, as well as the generation and recurrence of epileptic seizures and epileptic seizure-related neuronal damage. During the development of neurodegenerative diseases, overexpression of C1q, C1r, and C1s can be coupled with complement activation signals (e.g., β-amyloid, APP, cytokines (such as IFNγ, TNFα), etc.), also leading to inflammation.

[0293] By administering a pharmaceutical composition disclosed herein that inhibits complement activation, synapses that would otherwise be lost can be preserved.

[0294] Method for neutralizing complement bioactivity. The complement bioactivity affected can be (1) binding of C1q to autoantibodies, (2) binding of C1q to C1r, (3) binding of C1q to C1s, (4) binding of C1q to IgM, (5) binding of C1q to phosphatidylserine, (6) binding of C1q to pentraxin-3, (7) binding of C1q to C-reactive protein (CRP), (8) binding of C1q to globular C1q receptor (gC1qR), (9) binding of C1q to complement receptor 1 (CR1), (10) binding of C1q to beta-amyloid, (11) binding of C1q to calreticulin, (12) binding of C1q to apoptotic cells, or (13) binding of C1q to B cells. The affected complement biological activities can further be (1) activation of the classical complement activation pathway, (2) activation of antibodies and complement-dependent cytotoxicity, (3) CH50 hemolysis, (4) synaptic loss, (5) B cell antibody production, (6) dendritic cell maturation, (7) T cell proliferation, (8) cytokine production, (9) microglia activation, (10) Arthritis reaction, (11) phagocytosis of synapses or nerve terminals, and (12) activation of complement receptor 3 (CR3 / C3) expressing cells.

[0295] The method promotes improved maintenance of neuronal function in pathologies associated with synaptic loss. Maintenance of neural connections provides functional improvement in neurodegenerative diseases relative to untreated patients. Prevention of synaptic loss can include at least a measurable improvement, such as at least a 10% improvement in the number of synapses, at least a 20% improvement, at least a 50% improvement, or more, relative to a control group lacking such treatment, over a period of 1, 2, 3, 4, 5, 6 days, or at least a week.

[0296] In one aspect, described herein is a method of inhibiting synapse loss comprising administering a pharmaceutical composition disclosed herein to a patient suffering from adverse synapse loss. In some embodiments, the patient suffers from synapse loss due to a neurodegenerative disorder, a central nervous system disorder, or a peripheral nervous system disorder. In some embodiments, the method further comprises administering a neural progenitor cell or a neurogenesis enhancer. In some embodiments, the antibody binds to C1q and inhibits complement activation.

[0297] In one aspect, described herein is a method for treating or preventing a disease associated with complement activation in an individual in need of such treatment, the method comprising administering a pharmaceutical composition disclosed herein. The disease associated with complement activation can be a neurodegenerative disorder, which can be associated with synaptic loss or neural connection loss, associated with synaptic loss dependent on complement receptor 3 (CR3) / C3 or complement receptor CR1, associated with pathological activity-dependent synaptic pruning, or associated with synaptic phagocytosis of microglia. Neurodegenerative disorders can be Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, ophthalmic disorders, glaucoma, myotonic dystrophy, Guillain-Barre syndrome (GBS), myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease or Huntington's disease (HD). In some embodiments, the neurodegenerative disease is ALS, GBS or HD.

[0298] In some embodiments, the disease associated with complement activation is an inflammatory disease, an autoimmune disease, a complement-associated eye disease, or a metabolic disorder, such as diabetes, obesity, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), distal tissue damage after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardiac arrest-induced coronary endothelial dysfunction, type II membranous proliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, glaucoma, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degeneration, age-related macular degeneration (AMD), geographic atrophy, choroidal neovascularization (CNV), uveitis, diabetic retinopathy lesions, ischemia-related retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, neuromyelitis optica (NMO), central retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, ANCA vasculitis, Pursha retinopathy, Sjogren's dry eye disease, dry AMD, wet AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, multiple sclerosis, allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary disease (COPD), asthma, aspiration pneumonia, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis.

[0299] In some embodiments, the disease associated with complement activation is an autoimmune disease selected from the group consisting of myasthenia gravis, type 1 diabetes, Hashimoto's thyroiditis, Addison's disease, celiac disease, Crohn's disease, pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolytic anemia, tumor-associated syndromes, vasculitic diseases, hypocomplementemic urticarial vasculitis (HUV), polymyalgia rheumatica, temporal arteritis, Wegener's granulomatosis, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis.

[0300] In some embodiments, the disease associated with complement activation is a blood disorder selected from the group consisting of cold agglutinin hemolytic anemia (cold agglutinin disease), cold antibody hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, neonatal alloimmune thrombocytopenia, red blood cell count, Cellular alloimmunization, Felty's syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, systemic lupus erythematosus (SLE), glomerulonephritis, antiphospholipid antibody syndrome (APS), infection or drug-induced hematological disorders.

[0301] As used herein, "neurological" or "cognitive" function means that the reduction of synapses in the brain enhances the patient's thinking ability, function, etc. As used herein, the term "subject" covers mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class of mammals: humans; non-human primates, such as chimpanzees and other apes and monkey species; livestock, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs and cats; experimental animals, including rodents, such as rats, mice and guinea pigs, etc. The term does not indicate a particular age or sex.

[0302] The complement inhibitors of the present disclosure may be used in combination with any additional treatment, such as immunosuppressive therapy, for any of the diseases disclosed herein, including autoimmune diseases and / or neurodegenerative diseases, without limitation.

[0303] In some embodiments, the pharmaceutical compositions disclosed herein are co-administered with inhibitors of the alternative pathway of complement activation. Such inhibitors may include, but are not limited to, factor B blocking antibodies; factor D blocking antibodies; CD59, DAF, CR1, CR2, Crry or Comstatin-like peptides in the form of soluble, membrane-bound, labeled or fusion proteins that block C3 cleavage; non-peptide C3aR antagonists, such as SB 290157; cobra venom factor; or non-specific complement inhibitors, such as nafamostat mesilate (FUTHAN; FUT-175); aprotinin; K-76 monocarboxylic acid (MX-1); and heparin (see, for example, TEMollnes & M.Kirschfink, Molecular Immunology 43 (2006) 107–121). In some embodiments, the pharmaceutical compositions disclosed herein are co-administered with inhibitors of the interaction between autoantibodies and their own antigens. Such inhibitors may include purified soluble forms of autoantigens, or antigen mimetics, such as peptide or RNA derived mimotopes, including mimotopes of AQP4 antigens. Alternatively, such inhibitors may include blockers that recognize autoantigens and prevent autoantibody binding without triggering the classical complement pathway. Such blockers may include, for example, autoantigen binding RNA aptamers or antibodies that lack a functional C1q binding site in their Fc domain (e.g., Fab fragments or antibodies otherwise engineered to not bind C1q).

[0304] Example

[0305] Example 1: Formulation development of anti-C1q Fab fragment (Fab A)

[0306] The feasibility of high concentration anti-C1q antibody Fab fragment formulations was evaluated at 200 mg / mL using two formulation candidates: IVT-1 (10 mM histidine, 5% (w / v) trehalose dihydrate, 40 mM sodium chloride, pH 6.4) and IVT-2 (10 mM sodium succinate, 5% (w / v) trehalose dihydrate, 40 mM sodium chloride, 0.04% (w / v) polysorbate 20, pH 6.4). Subvisible particle (SVP) count data indicated that the presence of a surfactant was required to stabilize the anti-C1q antibody Fab fragment at 200 mg / mL.

[0307] Table 1 shows the subvisible particle counts for the two formulation candidates.

[0308]

[0309]

[0310] Polysorbate 20 (PS20) surfactant was added to all formulation candidates. Sorbitol and 2-hydroxypropyl-β-cyclodextrin (2HPBCD) were evaluated as alternatives to trehalose dihydrate. In addition, the effects of freeze / thaw (F / T) cycles (freezing at -20°C / thawing at room temperature), stirring stress, and accelerated stability were evaluated.

[0311] Table 2 shows the composition of the formulation candidates.

[0312]

[0313] At t=0, all samples were transparent with no visible particles.

[0314] Table 3 shows the properties of the formulation candidates at t=0.

[0315]

[0316]

[0317] After 5 freeze / thaw (F / T) cycles, the sample became turbid and did not comply with USP <789> In addition, the chemical stability of all formulation candidates remained unchanged after 5 F / T cycles.

[0318] Table 4 shows the properties after 5 freeze / thaw cycles.

[0319]

[0320]

[0321] Agitation showed a more severe stress state compared to freeze / thaw stress. After 5 days of agitation, all samples looked turbid with visible particles, which did not meet USP <789> SVP specifications. No change in chemical stability after stirring.

[0322] Table 5 shows the characteristics after stirring.

[0323]

[0324] Accelerated stability was checked at 8 weeks at 25° C. All samples except SVP met the specifications.

[0325] Table 6 shows the stability data at 25°C at 8 weeks.

[0326]

[0327] Accelerated stability was checked at 40°C at 8 weeks. All samples except SVP met specifications. Other notable but still acceptable changes included an increase in the percentage of pre- and inter-peak materials for cation exchange HPLC (CEX-HPLC) and high molecular weight materials (HMW) for SEC-HPLC.

[0328] Table 7 shows the stability data at 40°C at 8 weeks.

[0329]

[0330] After freeze / thaw cycles and agitation, all samples failed visual appearance and SVP, but passed all other product specifications.After 8 weeks of storage at 25°C and 40°C, all samples passed product specifications except SVP.

[0331] The surfactant level in the formulation and the visual appearance acceptance criteria (ie, no turbidity, no visible particles) were optimized after at least 3 freeze / thaw cycles and after storage at least 4 weeks at 25°C.

[0332] Subsequent studies focused on the SVP and visual appearance tests, which were shown to be the greatest indicators of stability compared to other tests for measuring changes after multiple freeze-thaw cycles and stirring stress.

[0333] The optimal surfactant concentration was determined to be in the range of 0.02% to 0.06%. In addition, polysorbate 20 (PS20) and polysorbate 80 (PS80) surfactants were compared at a fixed concentration of 0.06%. The study was performed at an anti-C1q antibody Fab fragment concentration of 100 mg / mL.

[0334] Table 8 shows the composition of the formulation candidates of 100 mg / mL anti-C1q antibody Fab fragments.

[0335]

[0336] Table 9 shows the properties of the formulation candidates at t=0.

[0337]

[0338]

[0339] There was no significant difference between PS20 and PS80. Based on the SVP results, a minimum surfactant (PS20) concentration of 0.04% (w / v) was required.

[0340] Table 10 shows the properties after freeze / thaw cycles and stirring.

[0341]

[0342] The effect of anti-C1q antibody Fab fragment concentration was evaluated at 100 mg / mL and 200 mg / mL in 0.06% (w / v) PS20. Reducing the protein concentration did not result in a corresponding reduction in the surfactant concentration.

[0343] Table 11 shows the effect of anti-C1q antibody Fab fragment concentration.

[0344]

[0345] Sodium succinate and sodium citrate buffers of varying pH were used to evaluate the effect of pH.

[0346] Table 12 shows the composition of the formulation candidates.

[0347]

[0348] The sodium succinate pH 5.5 sample turned into a gel when stored at 2°C-8°C. However, the gel state was reversible and turned into a liquid at room temperature. Both the sodium succinate and sodium citrate pH 6.4 samples went through this brief but distinct gel phase when thawed, but remained liquid at 2°C-8°C after complete thawing. However, all samples formulated at pH 7.2 did not exhibit any gelling behavior, transient or otherwise.

[0349] Table 13 shows the properties after freeze / thaw cycles and agitation.

[0350]

[0351] A pH-dependent gel phase transition was observed, showing a tendency for the product to gel as the pH decreased. In addition, no difference in SVP was observed between sodium succinate and sodium citrate at pH 7.2.

[0352] Anti-C1q antibody Fab fragments were formulated in 10 mM sodium citrate, 5% (w / v) trehalose dihydrate, 40 mM sodium chloride, 0.06% (w / v) polysorbate 20 (pH 7.2) and placed in a stable state.

[0353] Table 14 shows the stability of selected formulations at -70°C.

[0354]

[0355]

[0356] Table 15 shows the stability of selected formulations at 5°C.

[0357]

[0358] Table 16 shows the stability of selected formulations at 25°C.

[0359]

[0360]

[0361] Incorporated by Reference

[0362] Each of the patents, published patent applications, and non-patent references cited herein is hereby incorporated by reference in its entirety.

[0363] Equivalent solutions

[0364] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A pharmaceutical composition, comprising: (a) at least 75 mg / ml of anti-C1q antibody; (b) 5 mM to 25 mM sodium citrate or sodium succinate; (c) 3% to 7% trehalose; and (d) 0.04% to 0.06% polysorbate 20 or polysorbate 80, The pH of the pharmaceutical composition is 6.4-7.

5.

2. The pharmaceutical composition of claim 1, wherein the antibody comprises a light chain variable domain comprising HVR-L1 having an amino acid sequence of SEQ ID NO: 5, HVR-L2 having an amino acid sequence of SEQ ID NO: 6, and HVR-L3 having an amino acid sequence of SEQ ID NO:

7.

3. The pharmaceutical composition of any one of claims 1 to 2, wherein the antibody comprises a heavy chain variable domain comprising HVR-H1 having an amino acid sequence of SEQ ID NO: 9, HVR-H2 having an amino acid sequence of SEQ ID NO: 10, and HVR-H3 having an amino acid sequence of SEQ ID NO:

11.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a chimeric antibody, a multispecific antibody or an antibody derivative thereof.

5. The pharmaceutical composition of claim 4, wherein the antibody derivative is a one-armed antibody.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, and wherein the light chain variable domain comprises HVR-L1 having an amino acid sequence of SEQ ID NO: 5, HVR-L2 having amino acids of SEQ ID NO: 6, and HVR-L3 having amino acids of SEQ ID NO:

7.

7. The pharmaceutical composition of claim 6, wherein the light chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 35-38.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the antibody comprises a heavy chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 8 and 31-34, and wherein the heavy chain variable domain comprises HVR-H1 having an amino acid sequence of SEQ ID NO: 9, HVR-H2 having amino acids of SEQ ID NO: 10, and HVR-H3 having amino acids of SEQ ID NO:

11.

9. The pharmaceutical composition of claim 8, wherein the heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 31-34.

10. The pharmaceutical composition of any one of claims 1 to 4, wherein the antibody is an antibody fragment.

11. The pharmaceutical composition of claim 10, wherein the antibody fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a diabody or a single-chain antibody molecule.

12. The pharmaceutical composition of claim 11, wherein the antibody Fab fragment comprises the heavy chain Fab fragment of SEQ ID NO:

39.

13. The pharmaceutical composition of claim 11 or 12, wherein the antibody Fab fragment comprises the light chain Fab fragment of SEQ ID NO:

40.

14. The pharmaceutical composition of any one of claims 1 to 13, wherein the pharmaceutical composition comprises at least 100 mg / ml, at least 125 mg / ml, at least 150 mg / ml, at least 175 mg / ml, at least 200 mg / ml, at least 225 mg / ml or at least 250 mg / ml of the anti-C1q antibody.

15. The pharmaceutical composition of any one of claims 1 to 13, wherein the pharmaceutical composition comprises 75 mg / ml to 300 mg / ml, 100 mg / ml to 300 mg / ml, or 125 mg / ml to 250 mg / ml of the anti-C1q antibody.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the pharmaceutical composition comprises about 75 mg / ml, 100 mg / ml, 125 mg / ml, about 150 mg / ml, about 175 mg / ml, about 200 mg / ml, 225 mg / ml or about 250 mg / ml of an anti-C1q antibody Fab fragment.

17. A pharmaceutical composition as described in any one of claims 1 to 16, wherein the pharmaceutical composition comprises about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11mM, about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, about 21mM, about 22mM, about 23mM, about 24mM or about 25mM sodium citrate or sodium succinate.

18. The pharmaceutical composition of any one of claims 1 to 17, wherein the pharmaceutical composition is hypertonic or isotonic.

19. The pharmaceutical composition of any one of claims 1 to 18, wherein the pharmaceutical composition comprises about 3%, 4%, 5%, 6% or 7% trehalose.

20. The pharmaceutical composition of any one of claims 1 to 19, wherein the pharmaceutical composition comprises about 0.04%, 0.05%, or 0.06% polysorbate 20 or polysorbate 80.

21. The pharmaceutical composition of any one of claims 1 to 20, wherein the pH of the pharmaceutical composition is about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.

5.

22. The pharmaceutical composition of any one of claims 1 to 21, wherein the pharmaceutical composition further comprises 30 mM to 50 mM NaCl.

23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition comprises about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM NaCl.

24. The pharmaceutical composition of any one of claims 1 to 23, wherein the viscosity of the pharmaceutical composition is no more than 10 cP at 25°C.

25. The pharmaceutical composition of claim 24, wherein the viscosity of the pharmaceutical composition is about 1 cP, about 2 cP, about 3 cP, about 4 cP, about 5 cP, about 6 cP, about 7 cP, about 8 cP, about 9 cP, or about 10 cP at 25°C.

26. The pharmaceutical composition of any one of claims 1 to 25, wherein the pharmaceutical composition is stable for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 20 weeks, at least 21 weeks, at least 22 weeks, at least 23 weeks, or at least 24 weeks.

27. The pharmaceutical composition of any one of claims 1 to 26, wherein the pharmaceutical composition is stable for at least 1 year, at least 1.5 years, or at least two years.

28. The pharmaceutical composition of claim 26 or 27, wherein the pharmaceutical composition is stable at a temperature of -20°C to 25°C.

29. The pharmaceutical composition of claim 28, wherein the pharmaceutical composition is stable at a temperature of about -20°C, about -15°C, about -10°C, about -5°C, about 0°C, about 4°C, about 5°C, about 10°C, about 15°C, about 20°C, or about 25°C.

30. The pharmaceutical composition of claim 26, wherein the pharmaceutical composition is stable at a temperature greater than or equal to 25°C.

31. The pharmaceutical composition of claim 30, wherein the pharmaceutical composition is stable at a temperature between about 25°C and about 40°C.

32. The pharmaceutical composition of claim 30 or 31, wherein the pharmaceutical composition is stable at a temperature of about 25°C, about 30°C, about 35°C, or about 40°C.

33. A pharmaceutical composition as described in any one of claims 26 to 32, wherein the pharmaceutical composition is stable after at least one, at least two, at least three, at least four or at least five freeze / thaw cycles.

34. A pharmaceutical composition as described in any one of claims 26 to 33, wherein the pharmaceutical composition is stable after continuous stirring for at least one day, at least two days, or at least three days.

35. The pharmaceutical composition of any one of claims 1 to 34, wherein the pharmaceutical composition is suitable for intravitreal or intraocular injection.

36. The pharmaceutical composition of claim 35, wherein the pharmaceutical composition comprises 5 mM to 20 mM sodium citrate or sodium succinate.

37. The pharmaceutical composition of claim 36, wherein the pharmaceutical composition comprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM or about 20 mM sodium citrate or sodium succinate.

38. The pharmaceutical composition of any one of claims 35 to 37, wherein the pharmaceutical composition is contained in a syringe.

39. The pharmaceutical composition of any one of claims 35 to 37, wherein the pharmaceutical composition is pre-filled in a syringe.

40. The pharmaceutical composition of claim 38 or 39, wherein the syringe has a fill volume of no more than 300 microliters.

41. The pharmaceutical composition of any one of claims 38 to 40, wherein the syringe delivers a volume of 25 microliters to 100 microliters.

42. The pharmaceutical composition of any one of claims 38 to 41, wherein the syringe and / or the pharmaceutical composition is silicon-free and / or has a low level of particulates.

43. The pharmaceutical composition of any one of claims 38 to 42, wherein the syringe and / or the pharmaceutical composition has less than fifty particles per milliliter of 10 μm particles and / or less than five particles per milliliter of 25 μm particles.

44. The pharmaceutical composition of claim 43, wherein the syringe and / or the pharmaceutical composition complies with USP <789> Limit value.

45. A pharmaceutical composition as described in any one of claims 1 to 34, wherein the pharmaceutical composition is prepared for subcutaneous injection.

46. ​​The pharmaceutical composition of claim 45, wherein the pharmaceutical composition is contained in a syringe, an autoinjector, or a pen.

47. A pharmaceutical composition as claimed in claim 46, wherein the syringe, autoinjector or pen has a fill volume of no more than 10 ml.

48. A pharmaceutical composition as described in any one of claims 1 to 34, wherein the pharmaceutical composition is prepared for intravenous injection.

49. The pharmaceutical composition of claim 48, wherein the pharmaceutical composition is contained in an intravenous solution bag.

50. A syringe comprising the pharmaceutical composition of any one of claims 1 to 37.

51. The syringe of claim 50, wherein the syringe has a delivery volume of no more than 10 ml.

52. The syringe of any one of claims 50 to 51, wherein the syringe is an automatic reusable fixed dose pen.

53. The syringe of any one of claims 50 to 51, wherein the syringe is an automatic reusable variable dose pen.

54. The syringe of any one of claims 50 to 51, wherein the syringe is an automatic disposable fixed dose auto-injector.

55. The syringe of claim 50, wherein the syringe has a delivery volume of no more than 300 microliters.

56. The syringe of claim 50, wherein the syringe is a syringe.

57. A pre-filled syringe comprising the pharmaceutical composition of any one of claims 1 to 37.

58. The prefilled syringe of claim 57, wherein the prefilled syringe has a delivery volume of no more than 300 microliters.

59. The prefilled syringe of claim 57 or 58, wherein the prefilled syringe and / or the pharmaceutical composition is silicon-free and / or has low levels of particulates.

60. The prefilled syringe of any one of claims 57 to 59, wherein the prefilled syringe and / or the pharmaceutical composition has less than fifty particles per milliliter of 10 μm particles and / or less than five particles per milliliter of 25 μm particles.

61. The prefilled syringe of claim 60, wherein the prefilled syringe and / or the pharmaceutical composition complies with USP <789> Limit value.

62. A method of inhibiting synapse loss, the method comprising administering to a patient suffering from adverse synapse loss a pharmaceutical composition selected from any one of claims 1 to 49.

63. The method of claim 62, wherein the patient suffers from synaptic loss due to a neurodegenerative disorder, a central nervous system disorder, or a peripheral nervous system disorder.

64. The method of claim 63, wherein the neurodegenerative disorder is Guillain-Barre syndrome (GBS), amyotrophic lateral sclerosis (ALS), or Huntington's disease (HD).

65. The method of any one of claims 62 to 64, further comprising co-administering neural progenitor cells or a neurogenesis enhancer.

66. A method of treating or preventing a disease associated with complement activation in an individual in need of such treatment, the method comprising administering a pharmaceutical composition as claimed in any one of claims 1 to 49.

67. The method of claim 66, wherein the disease associated with complement activation is a neurodegenerative disorder.

68. The method of claim 67, wherein the neurodegenerative disorder is associated with synaptic loss or loss of neural connections.

69. The method of claim 67 or claim 68, wherein the neurodegenerative disorder is associated with synaptic loss dependent on complement receptor 3 (CR3) / C3 or complement receptor CR1.

70. The method of any one of claims 67 to 69, wherein the neurodegenerative disorder is associated with pathological activity-dependent synaptic pruning.

71. The method of any one of claims 67 to 70, wherein the neurodegenerative disorder is associated with synaptic phagocytosis by microglia.

72. The method of any one of claims 67 to 71, wherein the neurodegenerative disorder is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, an ophthalmic disorder, glaucoma, myotonic dystrophy, Guillain-Barre syndrome (GBS), myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down syndrome, Parkinson's disease, traumatic brain injury (TBI), epilepsy, or Huntington's disease (HD).

73. The method of claim 72, wherein the neurodegenerative disorder is amyotrophic lateral sclerosis (ALS).

74. The method of claim 72, wherein the neurodegenerative disorder is Huntington's disease.

75. The method of claim 66, wherein the disease associated with complement activation is an inflammatory disease, an autoimmune disease, a complement-associated eye disease, or a metabolic disorder.

76. The method of claim 75, wherein the inflammatory disease, autoimmune disease, complement-related eye disease, or metabolic disorder is selected from the group consisting of diabetes, obesity, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), distal tissue damage after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardiac arrest-induced coronary endothelial dysfunction, type II membranous proliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, glaucoma, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degeneration, age-related macular degeneration (AMD), geographic atrophy, choroidal neovascularization (CNV), uveitis, diabetic retinopathy , ischemia-related retinopathy, endophthalmitis, intraocular neovascularization, diabetic macular edema, pathological myopia, Hippel-Lindau disease, ocular histoplasmosis, neuromyelitis optica (NMO), central retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behcet's retinopathy, ischemic optic neuropathy, retinal vasculitis, ANCA vasculitis, Pursha retinopathy, Sjogren's dry eye, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary disease (COPD), asthma, aspiration pneumonia, multiple sclerosis, multifocal motor neuropathy (MMN), Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis.

77. The method of claim 76, wherein the disease associated with complement activation is an autoimmune disease selected from the group consisting of multifocal motor neuropathy (MMN), type 1 diabetes, Hashimoto's thyroiditis, Addison's disease, celiac disease, Crohn's disease, pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolytic anemia, tumor-associated syndromes, vasculitic diseases, hypocomplementemic urticarial vasculitis (HUV), polymyalgia rheumatica, temporal arteritis, Wegener's granulomatosis, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis.

78. The method of claim 76, wherein the disease associated with complement activation is a blood disorder selected from the group consisting of cold agglutinin hemolytic anemia (cold agglutinin disease), cold antibody hemolytic anemia, ABO incompatibility acute hemolytic reaction, warm agglutinin hemolytic anemia, warm antibody hemolytic anemia, warm autoimmune hemolytic anemia (WAIHA), autoimmune hemolytic anemia (AIHA), autoimmune thrombocytopenia, antiphospholipid syndrome, Evans syndrome, neonatal alloimmune platelet disease, Thrombocytopenia, red blood cell alloimmunization, Felty's syndrome, antibody-mediated thrombocytopenia, heparin-induced thrombocytopenia (HIT), heparin-induced thrombocytopenia and thrombosis (HITT), thrombotic thrombocytopenic purpura (TTP), immune thrombocytopenic purpura (ITP), thrombocytopenia, thrombosis, vasculitis, lupus nephritis, systemic lupus erythematosus (SLE), glomerulonephritis, antiphospholipid antibody syndrome (APS), infection or drug-induced hematological disorders.

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