Formulations for anti-C1Q antibodies

Abstract

The present disclosure generally relates to pharmaceutical compositions comprising anti-C1q antibodies, as well as methods of making and using such compositions. In certain embodiments, such compositions are highly concentrated anti-C1q antibody formulations that exhibit low viscosity and high stability, and therefore can be conveniently delivered to subjects in need thereof with reduced discomfort.

Description

[Technical Field]

[0001] Related Applications This patent application claims priority to U.S. Provisional Patent Application No. 63 / 414,206, filed October 7, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Therapeutic macromolecules (e.g., antibodies, antibody fragments, and antibody derivatives) must be formulated to not only render the molecules suitable for administration to patients but also maintain their stability during storage and subsequent use. For example, therapeutic antibodies in solution are susceptible to degradation, aggregation, or undesirable chemical modification unless the solution is properly formulated. The stability of antibodies in liquid formulations depends not only on the types of excipients used in the formulation but also on the amounts and ratios of the excipients relative to each other. Furthermore, when preparing liquid antibody formulations, other considerations besides stability must be taken into account. Examples of such additional considerations include the viscosity of the solution, the concentration of antibody that can be adjusted by a given formulation, and the visual quality or appearance of the formulation. Therefore, when formulating therapeutic antibodies, great care must be taken to arrive at a formulation that maintains stability, contains an appropriate concentration of antibody, and has a suitable viscosity, as well as other properties that allow the formulation to be conveniently administered to patients.

[0003] Anti-C1q antibodies are an example of therapeutically relevant macromolecules that require appropriate formulation. Anti-C1q antibodies are clinically useful in the treatment or prevention of diseases associated with complement activation, such as autoimmune, inflammatory, and neurodegenerative diseases, as well as cancer.

[0004] 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

[0005] In certain aspects, provided herein are pharmaceutical compositions comprising anti-C1q antibodies, e.g., full-length antibodies, antibody Fab fragments, and / or antibody derivatives, as well as methods of using and making such pharmaceutical compositions. In certain embodiments, the pharmaceutical compositions provided herein comprise highly concentrated anti-C1q antibody solutions (whether full-length antibodies, antibody Fab fragments, and / or antibody derivatives) while exhibiting high pharmaceutical stability and low viscosity. Thus, such compositions can be delivered to patients suffering from complement pathway-related diseases, for example, by intravitreal, subcutaneous, or intravenous injection. Furthermore, the high antibody concentration combined with the low viscosity of the compositions provided herein allows for smaller volumes of the compositions to be delivered to patients through smaller needles, reducing patient discomfort.

[0006] Provided herein is a pharmaceutical composition comprising at least 75 mg / ml of an anti-C1q antibody. The pharmaceutical composition may further comprise 5 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 pharmaceutical composition may have a pH of 6.4 to 7.5.

[0007] In some embodiments, the antibody comprises a light chain variable domain comprising HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the antibody comprises a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO: 11. The antibody can be a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antibody derivative thereof, e.g., a single-arm antibody.

[0008] In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% identity to an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, wherein the light chain variable domain comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence 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% identity to an amino acid sequence selected from SEQ ID NOs: 8 and 31-34, wherein the heavy chain variable domain comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO: 11. The heavy chain variable domain may comprise an amino acid sequence selected from SEQ ID NOs: 8 and 31-34.

[0009] In some embodiments, the antibody is an antibody fragment such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, or a single-chain antibody molecule. The antibody Fab fragment may preferably comprise the heavy chain Fab fragment of SEQ ID NO: 39. The antibody Fab fragment may preferably comprise the light chain Fab fragment of SEQ ID NO: 40.

[0010] 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 an anti-C1q antibody Fab fragment.

[0011] In some embodiments, 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, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM sodium citrate or sodium succinate. The pharmaceutical composition comprises 5 mM to 10 mM, 10 mM to 15 mM, 15 mM to 20 mM, or 20 mM to 25 mM sodium citrate or sodium succinate.

[0012] In some embodiments, the pharmaceutical composition is hypertonic or isotonic. In some embodiments, the pharmaceutical composition contains about 3%, 4%, 5%, 6%, or 7% trehalose. The pharmaceutical composition may contain 3% to 4%, 4% to 5%, 5% to 6%, or 6% to 7% trehalose. In some embodiments, the pharmaceutical composition contains about 0.04%, 0.05%, or 0.06% polysorbate 20 or polysorbate 80. The pharmaceutical composition may contain 0.04% to 0.05%, or 0.05% to 0.06% polysorbate 20 or polysorbate 80. In some embodiments, the pharmaceutical composition has a pH of 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 pharmaceutical composition may have a pH in the range of 6.0 to 6.5, or 6.5 to 7.0, or 7.0 to 7.5. In some embodiments, the pharmaceutical composition further comprises 30 mM to 50 mM NaCl. The pharmaceutical composition may comprise about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM NaCl. The pharmaceutical composition may comprise 30 mM to 35 mM, 35 mM to 40 mM, 40 mM to 45 mM, or 45 mM to 50 mM NaCl.

[0013] In some embodiments, the viscosity of the pharmaceutical composition is 10 cP or less at 25° C., e.g., 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 can be 1 cP to 5 cP or 5 cP to 10 cP at 25° C.

[0014] 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 may be stable for at least 1 year, at least 1.5 years, or at least 2 years. The pharmaceutical composition may be stable at temperatures between -20°C and 25°C, for example, 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 of 25°C or higher, for example, from about 25°C to about 40°C (e.g., about 25°C, about 30°C, about 35°C, or about 40°C).

[0015] In some embodiments, the pharmaceutical composition is stable after at least 1, at least 2, at least 3, at least 4, or at least 5 freeze / thaw cycles, hi some embodiments, the pharmaceutical composition is stable after at least 1 day, at least 2 days, or at least 3 days of continuous agitation.

[0016] In some embodiments, the pharmaceutical composition is suitable for intravitreal or intraocular injection. The pharmaceutical composition may comprise 5 mM to 20 mM sodium citrate or sodium succinate, for example, 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.

[0017] In some embodiments, the pharmaceutical composition is contained within a syringe. In some embodiments, the pharmaceutical composition is pre-filled within the syringe. The syringe can have a fill volume of 300 microliters or less. In some embodiments, the syringe is configured to deliver a volume of 25-100 microliters. In some embodiments, the syringe is silicone-free and / or has low particulate levels. In some embodiments, the pharmaceutical composition is silicone-free and / or has low particulate levels. In some embodiments, the pre-filled syringe has less than 50 particulates per ml of 10 μm particulates and / or less than 5 particulates per ml of 25 μm particulates. In some embodiments, the pharmaceutical composition has less than 50 particulates per ml of 10 μm particulates and / or less than 5 particulates per ml of 25 μm particulates. In some embodiments, the pre-filled syringe meets USP <789> In some embodiments, the pharmaceutical composition complies with the limits of U.S.P. <789> Complies with the restrictions of

[0018] In some embodiments, the pharmaceutical composition is prepared for subcutaneous injection. The pharmaceutical composition can be contained in a syringe, auto-injector, or pen. The syringe, auto-injector, or pen can have a fill volume of 10 ml or less.

[0019] In some embodiments, the pharmaceutical composition is prepared for intravenous injection. The pharmaceutical composition may be contained in an intravenous solution bag.

[0020] In one aspect, disclosed herein is a syringe containing the pharmaceutical composition disclosed herein. In some embodiments, the syringe has a delivery volume of 10 ml or less. 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 300 microliters or less. The syringe can be a syringe.

[0021] In another aspect, described herein are prefilled syringes comprising the pharmaceutical compositions described herein. In some embodiments, the prefilled syringes have a delivery volume of 300 microliters or less. In some embodiments, the prefilled syringes are silicone-free and / or have low particulate levels. In some embodiments, the pharmaceutical compositions are silicone-free and / or have low particulate levels. In some embodiments, the prefilled syringes have less than 50 particulates per ml for 10 μm particulates and / or less than 5 particulates per ml for 25 μm particulates. In some embodiments, the prefilled syringes have a particle size of less than USP 100 μm. <789> In some embodiments, the pharmaceutical composition complies with the limits of U.S.P. <789> Complies with the restrictions of

[0022] In yet another aspect, described herein is a method for inhibiting synapse loss. The method may include administering a pharmaceutical composition described herein to a patient suffering from adverse synapse loss. In some embodiments, the patient suffers from synapse loss as a result of a neurodegenerative disorder, a central nervous system disorder, or a peripheral nervous system disorder. The neurodegenerative disorder may be Guillain-Barré syndrome (GBS), amyotrophic lateral sclerosis (ALS), or Huntington's disease (HD).

[0023] In one aspect, disclosed herein is a method for treating or preventing a disease associated with complement activation in an individual in need thereof. The method may include administering a pharmaceutical composition described herein. The disease associated with complement activation may be a neurodegenerative disorder, which may be associated with synaptic loss or loss of neural connections, synaptic loss dependent on complement receptor 3 (CR3) / C3 or complement receptor CR1, pathological activity-dependent synaptic pruning, or synaptic phagocytosis by microglia. In some embodiments, the neurodegenerative disorder is Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, ophthalmic disorders, glaucoma, myotonic dystrophy, Guillain-Barré syndrome (GBS), myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down's 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.

[0024] 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. Examples of the inflammatory disease, autoimmune disease, complement-associated eye disease, or metabolic disorder include diabetes, obesity, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), remote tissue injury after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, glaucoma, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degenerative diseases, age-related macular degeneration (AMD), geographic atrophy, choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, intraocular neoplasia, and the like. The condition may be selected from biovascular disease, diabetic macular edema, pathologic myopia, von 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, Purcher's retinopathy, Sjogren's dry eye disease, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome (COPD), asthma, aspiration pneumonia, multiple sclerosis, multifocal motor neuropathy (MMN), Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis.

[0025] The disease associated with complement activation can be an autoimmune disease selected from 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, paraneoplastic 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.

[0026] Diseases associated with complement activation include cold agglutinin hemolytic anemia (cold agglutinin disease), cold antibody hemolytic anemia, ABO incompatible 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 alloimmunization, Felty syndrome, and antibody-mediated hemolysis. The blood disorder may be selected from 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 syndrome (APS), infection, or a drug-induced hematological disorder. DETAILED DESCRIPTION OF THE INVENTION

[0027] overview Provided herein are novel pharmaceutical compositions comprising antibodies, antibody fragments, or antibody derivatives, particularly antibody Fab fragments, capable of binding to C1q, as well as methods of making and using such pharmaceutical compositions. Also provided herein are uses of the pharmaceutical compositions for preventing, reducing the risk of developing, or treating diseases associated with complement activation.

[0028] definition As used herein, "a" or "an" can mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising," the terms "a" or "an" can mean one or more. For example, a reference to an "antibody" is a reference to one to many antibodies. As used herein, "another" can mean at least a second or more.

[0029] The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The term "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, antibody fragments, and antibody derivatives, so long as they exhibit biological activity. The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. H and V L When these heavy chains pair together, they form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th Ed., Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6. Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domain. Depending on the amino acid sequence of the constant domain (CH) of their heavy chain, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM (having heavy chains designated alpha ("α"), delta ("δ"), epsilon ("ε"), gamma ("γ"), and mu ("μ"), respectively). The gamma and alpha classes are further divided into subclasses (isotypes) based on relatively minor differences in CH sequence and function; for example, 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, for example, in Abbas et al., Cellular and Molecular Immunology, 4 th This is described in ed. (WBSaunders Co., 2000).

[0030] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy and light chains are respectively referred to as "V H " and "V L " These domains are generally the most variable parts of an antibody (relative to other antibodies of the same class) and contain the antigen-binding site. The variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy and light chain variable domains each contain four FR regions that largely adopt a beta-sheet configuration connected by three HVRs that form loops that connect, and in some cases form part of, the beta-sheet structure. The HVRs in each chain are held in close proximity and connected to each other by the FR regions, and together with the HVRs of the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institutes of Health, Bethesda, MD (1991)). The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity.

[0031] As used herein, the term "CDR" or "complementarity-determining region" is intended to mean the noncontiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. CDRs are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. 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) (the definitions include overlapping or subsets of amino acid residues when compared against each other). Nevertheless, application of either definition to refer to the CDRs of an antibody or grafted antibody or variants thereof is intended to be within the scope of the terms defined and used herein. As used herein, the terms "CDR-L1," "CDR-L2," and "CDR-L3" refer to the first, second, and third CDRs, respectively, in a light chain variable region. As used herein, the terms "CDR-H1," "CDR-H2," and "CDR-H3" refer to the first, second, and third CDRs, respectively, in a heavy chain variable region. As used herein, the terms "CDR-1," "CDR-2," and "CDR-3" refer to the first, second, and third CDRs, respectively, in the variable region of either chain.

[0032] Several HVR and CDR delineations are used and encompassed herein. Kabat complementarity-determining regions (CDRs), HVRs, are based on sequence variability and are the most commonly used (Kabat et al., supra). Chothia, instead, refer to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). AbM HVRs represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are described below. Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H32 H30-H35B (Kabat numbering) H1 H31-H35 H26-H35 H26-H32 H30-H35 (Chothia numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58 H3 H95-H102 H95-H102 H96-H101 H93-H101

[0033] HVRs may include "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65 or 49-65 (preferred embodiment) (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these extended HVR definitions.

[0034] The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise stated herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system. Unless otherwise stated herein, references to residue numbers in the constant domain of an antibody refer to residue numbering according to the EU numbering system (see, e.g., U.S. Patent Publication No. 2010-280227).

[0035] As used herein, the term "C1q antibody" should be interpreted similarly to "anti-C1q antibody" and refers to 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.

[0036] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies of the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0037] 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 the intact antibody or the F region of the antibody that retains or has altered 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)). Additional examples of antibody fragments include antibody derivatives, such as single-chain antibody molecules formed from antibody fragments, monovalent antibodies, and multispecific antibodies.

[0038] An "antibody derivative" is any construct that contains the antigen-binding region of an antibody. Examples of antibody derivatives include single-chain antibody molecules, single-arm antibodies, antibodies with a single antigen-binding arm, univalent antibodies, and multispecific antibodies formed from antibody fragments.

[0039] The term "single-arm antibody" as used herein encompasses 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 first and second Fc polypeptides. In some embodiments, one of the Fc polypeptides, but not both, is an N-terminally truncated heavy chain. In some embodiments, the N-terminally truncated heavy chain lacks the heavy chain variable domain, and preferably the N-terminally truncated heavy chain lacks heavy chain constant domain 1. In some embodiments, the antibody may be a bivalent antibody, with one arm binding to C1q and the other arm binding to a different antigen. Such antibodies do not crosslink and activate C1q in response to other antigens. A single-arm antibody may have a single antigen-binding arm. As used herein, "an antibody with a single antigen-binding arm" refers to 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 inert antigen-binding arm that is incapable of binding to the antigen, or 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.

[0040] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments and one residual "Fc" fragment, a designation reflecting its ability to crystallize readily. The Fab fragment contains the entire light chain plus the variable region domain of the heavy chain (V H ), and the first constant domain of one heavy chain (C H 1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking antigen. The Fab' fragment is C HF(ab')2 antibody fragments differ from Fab fragments by having several additional residues at the carboxy terminus of one domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0041] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody are determined by sequences in the Fc region, which is also recognized by Fc receptors (FcRs) found on certain cell types.

[0042] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) may be removed, for example, during antibody production or purification, or by recombinantly engineering the nucleic acid encoding the antibody heavy chain. Thus, intact antibody compositions may include antibody populations with all K447 residues removed, antibody populations lacking the removed K447 residue, and antibody populations having a mixture of antibodies with and without the K447 residue. Native-sequence Fc regions suitable for use in the antibodies of the present disclosure include human IgG1, IgG2, IgG3, and IgG4.

[0043] A "native sequence Fc region" comprises an amino acid sequence identical to that 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.

[0044] A "variant Fc region" comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Preferably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or the Fc region of a parent polypeptide, e.g., about one to about ten amino acid substitutions, and preferably about one to about five amino acid substitutions, in the native-sequence Fc region or in the Fc region of the parent polypeptide. A variant Fc region herein preferably retains at least about 80% homology with a native-sequence Fc region and / or the Fc region of a parent polypeptide, and most preferably at least about 90% homology thereto, and more preferably at least about 95% homology thereto.

[0045] As used herein, "chimeric antibody" refers to antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). Chimeric antibodies of interest herein include PRIMATIZED® antibodies, in which the antigen-binding region of the antibody is derived from an antibody generated, for example, by immunizing macaque monkeys with the antigen of interest. As used herein, "humanized antibodies" are a subset of "chimeric antibodies."

[0046] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In some embodiments, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and / or capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications may be made to further refine antibody performance, such as binding affinity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence and all or substantially all of the FR regions correspond to those of a human immunoglobulin sequence, although the FR regions may include one or more individual FR residue substitutions which improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FRs typically will not exceed six in the H chain and not exceed three in the L chain. The humanized antibody also optionally will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further 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. Patent Nos. 6,982,321 and 7,087,409.

[0047] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of 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). The methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomies, that have been engineered to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). See also Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies produced by human B-cell hybridoma technology.

[0048] An "acceptor human framework," as used herein, is a framework that comprises the amino acid sequence of a VL framework 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 the same amino acid sequence as it, 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. If pre-existing amino acid changes are present in the VH, preferably those changes are present at only three, two, or one of positions 71H, 73H, and 78H, e.g., the amino acid residues at those positions may be 71A, 73T, and / or 78A. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0049] A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in the selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is made from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include, for VL, the subgroup can be subgroup kappa I, kappa II, kappa III, or kappa IV as in Kabat et al., supra. Additionally, for VH, the subgroup can be subgroup I, subgroup II, or subgroup III as in Kabat et al., supra.

[0050] An "amino acid modification" at a specified position refers to a substitution or deletion of the specified residue, or an insertion of at least one amino acid residue adjacent to the specified residue. An insertion "adjacent to" a specified residue means an insertion within one to two residues thereof. The insertion may be on the N-terminal or C-terminal side of the specified residue. A preferred amino acid modification herein is a substitution.

[0051] "Identity," as used herein, indicates that the amino acid residue at any particular position in the aligned sequences is the same between the sequences. "Similarity," as used herein, indicates that the amino acid residue at any particular position in the aligned sequences is of a similar type between the sequences. For example, leucine can be substituted for isoleucine or valine. Other amino acids that can often be substituted for one another include: - phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains); - lysine, arginine and histidine (amino acids with basic side chains); - aspartate and glutamate (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and - including but not limited to cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0052] 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, eds., 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., eds., M Stockton Press, New York, 1991).

[0053] As used herein, an "interaction" between C1q and a second protein includes, but is not limited to, protein-protein interactions, physical interactions, chemical interactions, bonds, covalent bonds, and ionic bonds. As used herein, an antibody "inhibits the interaction" between two proteins if the antibody disrupts, reduces, or completely eliminates the interaction between the two proteins. An antibody of the present disclosure, or a fragment thereof, "inhibits the interaction" between two proteins if the antibody or a fragment thereof binds to one of the two proteins.

[0054] As used herein, with respect to peptide, polypeptide, or antibody sequences, "percent (%) amino acid sequence identity" and "homology" refer to the percentage of amino acid residues in a candidate sequence that are identical to those in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent 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™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art, necessary to achieve maximal alignment over the entire length of the sequences being compared.

[0055] The term "preventing," when used in reference to a condition, is art-recognized and well understood in the art, and includes administration of a composition that reduces the frequency of or delays the onset of symptoms of a medical condition in a subject compared to subjects not administered the composition. Thus, for example, preventing a disease associated with complement activation includes, for example, reducing the occurrence of a disease associated with complement activation in a population of patients receiving prophylactic treatment compared to an untreated control population by a statistically and / or clinically significant amount, and / or delaying the onset of a disease associated with complement activation in a treated population relative to an untreated control population.

[0056] As used herein, the terms "specifically recognize" or "specifically bind" refer to a measurable and reproducible interaction, such as attraction or binding, between a target and an antibody that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules. For example, an antibody that specifically or preferentially binds to a target or epitope is one that binds to this target or epitope with higher affinity, avidity, more readily, and / or with a longer duration than it binds to other targets or other epitopes of targets. For example, it is understood that an antibody (or moiety) 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 exclusive binding (although it can include exclusive binding). An antibody that specifically binds to a target has at least about 10 3 M -1 or 10 4 M -1 , sometimes about 10 5 M -1 or 10 6 M -1 , and in other cases, about 10 6 M -1 or 10 7 M -1 , about 10 8 M -1 ~10 9 M -1 , or about 10 10 M -1 ~10 11 M -1 The antibody may have a binding constant of 100 uA or higher. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0057] The term "subject," as used herein, refers to a living mammal and may be used interchangeably with the term "patient." Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates, e.g., chimpanzees, and other ape and monkey species; farm animals, e.g., cows, horses, sheep, goats, pigs; domestic animals, e.g., rabbits, dogs, and cats; laboratory animals, including rodents, e.g., rats, mice, and guinea pigs; and the like. The term does not denote a particular age or sex.

[0058] The term "therapeutically effective amount" of a compound in the context of a subject treatment method refers to the amount of compound(s) in a preparation that, when administered as part of a desired dosing regimen (to a mammal, preferably a human), will treat a disorder or condition, for example, in accordance with clinically accepted standards for cosmetic purposes, reduce symptoms, ameliorate disease, or delay the onset of a disease condition, at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutically effective amount herein may vary depending on factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit a desired response in the individual.

[0059] As used herein, the term "treating" or "treatment" includes reducing, arresting, or reversing the symptoms, clinical signs, or underlying pathology of a condition to stabilize or improve a subject's condition, or to reduce the likelihood that the subject's condition will worsen to the same extent as if the subject had not received treatment.

[0060] antibody All sequences referred to in this disclosure are incorporated by reference from 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 (each of which is incorporated by reference herein for the antibodies and related compositions it discloses).

[0061] Antibodies can be prepared using recombinant DNA engineering techniques. Such modified versions include, for example, those created from native antibody variable regions by insertions, deletions, or changes within or to the amino acid sequence of the native antibody. Particular examples of this type include those engineered variable region domains containing at least one CDR from one antibody and optionally one or more framework amino acids and the remainder of the variable region domain from a second antibody. DNA encoding an antibody can be prepared by deleting all but the desired portion of the DNA encoding the antibody. DNA encoding a chimerized antibody can be prepared by recombining DNA encoding substantially or exclusively human constant regions with DNA encoding variable regions substantially or exclusively derived from variable region sequences of a mammal other than a human. DNA encoding a humanized antibody can be prepared by recombining DNA encoding variable regions other than the constant regions and complementarity-determining regions (CDRs) substantially or exclusively derived from corresponding human antibody regions with DNA encoding CDRs substantially or exclusively derived from a mammal other than a human.

[0062] Suitable sources of DNA molecules encoding antibodies include cells, such as hybridomas, that express full-length antibodies. For example, antibodies can be isolated from host cells that express expression vectors encoding the heavy and / or light chains of the antibody.

[0063] Antibody fragments, including but not limited to Fab fragments, and / or antibody derivatives can also be prepared by using recombinant DNA modification techniques, including the manipulation and re-expression of DNA encoding antibody variable and constant regions. Standard molecular biology techniques can be used to modify, add, or delete additional amino acids or domains, as desired. Any changes to the variable or constant regions are still encompassed by the terms "variable" and "constant" regions as used herein. In some instances, C H C so that translation of the 1 domain stops at the interchain cysteine. H PCR is used to generate antibody fragments by introducing a stop codon immediately after the codon encoding the interchain cysteine ​​of antibody C. Methods for designing suitable PCR primers are well known in the art and are described in detail in the literature for antibody C. H The sequence of one domain is readily available. In some embodiments, a stop codon can be introduced using site-directed mutagenesis techniques.

[0064] Antibodies of the present disclosure may 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 antibody heavy and light chains are derived from IgG. The antibody heavy and / or light chains may be derived from mouse IgG or human IgG. In certain other preferred embodiments, the antibody heavy and / or light chains are derived from human IgG1. In yet other preferred embodiments, the antibody heavy and / or light chains are derived from human IgG4.

[0065] The antibodies of the present disclosure can bind to and inhibit the biological activity of C1q, C1r, or C1s, for example, (1) C1q binding to autoantibodies, (2) C1q binding to C1r, (3) C1q binding to C1s, (4) C1q binding to phosphatidylserine, (5) C1q binding to pentraxin-3, (6) C1q binding to C-reactive protein (CRP), (7) C1q binding to globular C1q receptor (gC1qR), (8) C1q binding to complement receptor 1 (CR1), (9) C1q binding to B-amyloid, or (10) C1q binding to calreticulin.In other embodiments, the biological activity of C1q is (1) activation of the classical complement pathway, (2) reduced lysis and / or reduced C3 deposition, (3) activation of antibody and complement dependent cytotoxicity, (4) CH50 hemolysis, (5) reduced erythrocyte lysis, (6) reduced erythrophagocytosis, (7) reduced dendritic cell infiltration, (8) inhibition of complement mediated erythrocyte lysis, (9) reduced lymphocyte infiltration, (10) reduced macrophage infiltration, (11) reduced antibody deposition, (12) reduced erythrocyte lysis, (13) reduced erythrocyte phagocytosis, (14) reduced dendritic cell infiltration, (15) reduced erythrocyte lysis, (16) reduced erythrocyte lysis, (17) reduced lymphocyte infiltration, (18) reduced macrophage infiltration, (19) reduced antibody deposition, (20) reduced erythrocyte lysis, (21) reduced erythrocyte lysis, (22) reduced erythrocyte lysis, (23) reduced erythrocyte lysis, (24) reduced erythrocyte lysis, (25) reduced erythrocyte lysis, (26) reduced erythrocyte phagocytosis, (27) reduced dendritic cell infiltration, (28) reduced erythrocyte lysis, (29) reduced lymphocyte infiltration, (30) reduced macrophage infiltration, (31) reduced erythrocyte lysis, (32) reduced erythrocyte lysis, (33) reduced erythrocyte lysis, (34) reduced erythrocyte lysis, (35) reduced erythrocyte lysis, (36) reduced erythrocyte lysis, (37) reduced erythrocyte lysis, (38) reduced erythrocyte lysis, (39) reduced erythrocyte lysis, (40) reduced erythrocyte ) reduced neutrophil infiltration, (13) reduced platelet phagocytosis, (14) reduced platelet lysis, (15) improved graft survival, (16) reduced macrophage-mediated phagocytosis, (17) reduced autoantibody-mediated complement activation, (18) reduced red blood cell destruction due to transfusion reactions, (19) reduced alloantibody-mediated red blood cell lysis, (20) reduced hemolysis due to transfusion reactions, (21) reduced alloantibody-mediated platelet lysis, (22) improved anemia, (23) reduced eosinophilia, (24) 4) Decreased C3 deposition on red blood cells (e.g., decreased C3b, iC3b, etc. deposition on RBCs), (25) Decreased C3 deposition on platelets (e.g., decreased C3b, iC3b, etc. deposition on platelets), (26) Decreased anaphylatoxin production, (27) Decreased autoantibody-mediated rash formation, (28) Decreased autoantibody-induced lupus erythematosus, (29) Decreased red blood cell destruction in transfusion reactions, (30) Decreased platelet lysis in transfusion reactions, (31) Decreased fertility These include decreased mast cell activation, (32) decreased mast cell histamine release, (33) decreased vascular permeability, (34) decreased complement deposition on graft endothelium, (35) B cell antibody production, (36) dendritic cell maturation, (37) T cell proliferation, (38) cytokine production, (39) microglial activation, (40) Arthus reaction, (41) decreased anaphylatoxin production in graft endothelium, or (42) activation of complement receptor 3 (CR3 / C3)-expressing cells.

[0066] In some embodiments, the 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 may also be 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.

[0067] Other in vitro assays for measuring complement activity include ELISA assays for measuring the cleavage products of complement components or complexes formed during complement activation. Complement activation via the classical pathway can be measured by monitoring serum C4d and C4 levels. Activation of the alternative pathway can be measured with ELISA by assessing the levels of Bb or C3bBbP complexes in the circulation. In vitro antibody-mediated complement activation assays can also be used to assess inhibition of C3a generation.

[0068] 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 single-arm antibodies. In some embodiments, the antibody is a humanized antibody.

[0069] An antibody of the present disclosure may also be an antibody fragment, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, a diabody, or a single-chain antibody molecule. In some embodiments, the antibody fragment is a Fab fragment.

[0070] In some embodiments, the antibody is a human monoclonal antibody that may be prepared, expressed, produced, or isolated by recombinant means, such as (a) an antibody isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) an antibody isolated from a host cell transformed to express the antibody, e.g., an antibody isolated 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, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), thus modifying the V and constant regions of the recombinant antibody. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0071] In some embodiments, the antibodies are humanized and / or chimeric monoclonal antibodies that can be established by immunizing rodents (e.g., mice, rats, hamsters, and guinea pigs) with (1) native 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 either eukaryotic or prokaryotic systems, or derived fragments thereof. Other animals, such as non-human primates, transgenic mice expressing human immunoglobulins, and severe combined immunodeficient (SCID) mice engrafted with human B lymphocytes, can be used for immunization.

[0072] Polyclonal and monoclonal antibodies are naturally produced as immunoglobulin (Ig) molecules in the immune system's response to pathogens. The predominant form, the approximately 150 kDa IgG1 molecule, which has a concentration of 8 mg / ml in human serum, is composed of two identical approximately 50 kDa heavy chains and two identical approximately 25 kDa light chains.

[0073] Hybridomas can be generated by conventional procedures by fusing B lymphocytes from immunized animals with myeloma cells. Anti-C1q antibodies can also be generated 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.

[0074] The inhibitory activity of the antibodies identified in the screening process against complement activation can be evaluated 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 specific inhibitory activity against the classical complement pathway are cloned by limiting dilution. The antibodies are purified for characterization of their specificity for human C1q by the assay described above.

[0075] Anti-complement C1q antibodies The anti-C1q antibodies disclosed herein are potent inhibitors of C1q and can be administered for continuous inhibition of C1q function for any period of time, and then optionally discontinued to allow restoration of normal C1q function at a time when its activity may be important.

[0076] C1q is a large 460 kDa multimeric protein consisting of 18 polypeptide chains (six C1q A chains, six C1q B chains, and six C1q C chains). The C1r and C1s complement proteins bind to the C1q tail region to form the C1 complex (C1qr2s2).

[0077] The antibodies of the present disclosure specifically recognize complement factor C1q and / or C1q in the C1 complex of the classical complement activation pathway. The bound complement factor can be from any organism that has a complement system, including, but not limited to, any mammalian organism, such as a human, mouse, rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pig.

[0078] As used herein, a "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., C1qr 2 s 2 ) refers to

[0079] The anti-C1q antibodies disclosed herein can inhibit C1 complex formation.

[0080] As used herein, "complement factor C1q" refers to both the wild-type sequence and the naturally occurring variant sequence.

[0081] A non-limiting example of a complement factor C1q recognized by the antibodies of the present disclosure is human C1q, which comprises three polypeptide chains A, B, and C: C1q, chain A (Homo sapiens), accession number protein Database: NP_057075.1; GenBank number: NM_015991: >gi|7705753|ref|NP_057075.1|Complement C1q Subcomponent Subunit A Precursor [Homo sapiens] (SEQ ID NO: 1) MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAIRRN PPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYYFTFQVLSQWEICLSIVSSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA. C1q, chain B (Homo sapiens), accession number protein Database: NP_000482.3; GenBank number: NM_000491.3: >gi|87298828|ref|NP_000482.3|Complement Clq Subcomponent Subunit B Precursor [Homo sapiens] (SEQ ID NO: 23) MMMKIPWGSIPVLMLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSAT RTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA. C1q, chain C (Homo sapiens), accession number protein Database: NP_001107573.1; GenBank number: NM_001114101.1: >gi|166235903|ref|NP_001107573.1|Complement C1q Subcomponent Subunit C Precursor [Homo sapiens] (SEQ ID NO: 24) MDVGPSSLPHLGLKLLLLLLLLPLRGQANTGCYGIPGMPGLPGAPGKDGYDGLPGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPGMPGVPGPMGIPGEPGEEGRYKQKFQSVFT VTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD.

[0082] Thus, anti-C1q antibodies of the present disclosure may bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of the C1q protein. In some embodiments, anti-C1q antibodies of the present disclosure bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of human C1q or its homologues, e.g., 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.

[0083] All sequences referred to in the following 20 paragraphs are incorporated by reference from US Pat. No. 9,708,394, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0084] Light and heavy chain variable domain sequences of antibody M1 (Mab1) (incorporated by reference from U.S. Pat. No. 9,708,394) The nucleic acid and amino acid sequences encoding the light chain variable and heavy chain variable domains of antibody M1 were determined using standard techniques. The amino acid sequence of the light chain variable domain of antibody M1 is as follows:

[0085] TIFF2025533847000001.tif17170

[0086] The hypervariable regions (HVRs) of the light chain variable domains are shown 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).

[0087] The amino acid sequence of the heavy chain variable domain of antibody M1 is:

[0088] TIFF2025533847000002.tif22170

[0089] The hypervariable regions (HVRs) of the heavy chain variable domains are shown in bold and underlined text. In some embodiments, HVR-H1 of the M1 heavy chain variable domain has the sequence GYHFTSYWMH (SEQ ID NO:9), HVR-H2 of the M1 heavy chain variable domain has the sequence VIHPNSGSINYNEKFES (SEQ ID NO:10), and HVR-H3 of the M1 heavy chain variable domain has the sequence ERDSTEVLPMDY (SEQ ID NO:11).

[0090] The nucleic acid sequence encoding the light chain variable domain was determined to be: GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAACAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTT TGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAACATAATGAATACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 12).

[0091] The nucleic acid sequence encoding the heavy chain variable domain was determined to be: CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGTCTTCTGGCTACCATTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGTGATTCATCCTAATAGTGGTAGTATTAACTACAATGAG AAGTTCGAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCGGCGGTCTATTATTGTGCAGGAGAGAGAGATTCTACGGAGGTTCTCCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 13).

[0092] Mab1-Fab is the Fab of the Mab1 (M1) antibody.

[0093] Deposit of materials The following materials have been deposited in accordance with the Budapest Treaty with the American Type Culture Collection, ATCC Patent Depository, 10801 University Blvd., Manassas, Va. 20110-2209, USA (ATCC): [Table 1]

[0094] A hybridoma cell line (murine hybridoma C1qM1 7788-1(M)051613) producing the M1 antibody has been deposited with the ATCC under conditions ensuring that cultures will be available during the pendency of a patent application and for 30 years, or five years after the latest claim, or the life of the patent, whichever is longer. The deposit will be replaced if it becomes non-viable during that period. The deposit will be available as required by foreign patent laws in countries in which counterpart applications to this application, or their progeny, are filed. However, it should be understood that the availability of the deposit does not constitute a license to practice the invention in derogation of patent rights granted by government action.

[0095] Humanized Anti-Complement C1q Antibody (Incorporated by reference from U.S. Pat. No. 10,316,081) The humanized antibodies of the present disclosure specifically bind to complement factor C1q and / or C1q protein in the C1 complex of the classical complement pathway. The humanized anti-C1q antibodies may specifically bind to human C1q, human and mouse C1q, rat C1q, or human C1q, mouse C1q, and rat C1q.

[0096] 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 an amino acid sequence having at least 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90% homology to SEQ ID NO: 26. The human IgG4 heavy chain constant region may comprise an Fc region with one or more modifications and / or amino acid substitutions according to Kabat numbering. In such cases, the Fc region comprises a leucine to glutamate amino acid substitution at position 248, which inhibits the Fc region from interacting with an Fc receptor. In some embodiments, the Fc region comprises a serine to proline amino acid substitution at position 241, which prevents arm switching in the antibody.

[0097] Human IgG4(S241P L248E; corresponding to S108P and L115E of SEQ ID NO: 26) is the amino acid sequence of the heavy chain constant domain: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 26).

[0098] 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% identity 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% identity to an amino acid sequence selected from any one of SEQ ID NOs: 35-38.

[0099] The amino acid sequence of the heavy chain variable domain variant 1 (VH1) is:

[0100] TIFF2025533847000004.tif22170The hypervariable regions (HVRs) of VH1 are shown in bold and underlined text.

[0101] The amino acid sequence of the heavy chain variable domain variant 2 (VH2) is:

[0102] TIFF2025533847000005.tif22170The hypervariable regions (HVRs) of VH2 are shown in bold and underlined text.

[0103] The amino acid sequence of the heavy chain variable domain variant 3 (VH3) is:

[0104] TIFF2025533847000006.tif22170The hypervariable regions (HVRs) of VH3 are shown in bold and underlined text.

[0105] The amino acid sequence of the heavy chain variable domain variant 4 (VH4) is:

[0106] TIFF2025533847000007.tif22170The hypervariable regions (HVRs) of VH4 are shown in bold and underlined text.

[0107] The amino acid sequence of the kappa light chain variable domain variant 1 (Vκ1) is:

[0108] TIFF2025533847000008.tif17170The hypervariable regions (HVRs) of Vκ1 are shown in bold and underlined text.

[0109] The amino acid sequence of the kappa light chain variable domain variant 2 (Vκ2) is:

[0110] TIFF2025533847000009.tif17170The hypervariable regions (HVRs) of Vκ2 are shown in bold and underlined text.

[0111] The amino acid sequence of the kappa light chain variable domain variant 3 (Vκ3) is:

[0112] TIFF2025533847000010.tif17170The hypervariable regions (HVRs) of Vκ3 are shown in bold and underlined text.

[0113] The amino acid sequence of the kappa light chain variable domain variant 4 (Vκ4) is:

[0114] TIFF2025533847000011.tif17170The hypervariable regions (HVRs) of Vκ4 are shown in bold and underlined text.

[0115] The antibody may comprise a light chain variable domain amino acid sequence 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 QQHNEYPLT (SEQ ID NO: 7). The antibody may comprise a heavy chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NOs: 31-34, while preferably retaining HVR-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO: 10), and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0116] 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.

[0117] The full-length antibody Mab2 contains a heavy chain variable domain variant 3 (VH3) (SEQ ID NO: 33) and a kappa light chain variable domain variant 3 (Vκ3) (SEQ ID NO: 37). Mab2-Fab is the Fab of the Mab2 antibody.

[0118] The antibody can 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.

[0119] The amino acid sequence of the heavy chain is:

[0120] TIFF2025533847000012.tif49170The hypervariable regions (HVRs) of VH3 are shown in bold and underlined text.

[0121] The amino acid sequence of the light chain is:

[0122] TIFF2025533847000013.tif27170

[0123] The complementarity determining regions (CDRs) of SEQ ID NO: 40 are shown in bold and underlined text.

[0124] In some embodiments, a humanized anti-C1q antibody of the present disclosure comprises a heavy chain variable region comprising a Fab region and a heavy chain constant region comprising an Fc region, wherein the Fab region specifically binds to a C1q protein of the present disclosure, while the Fc region is incapable of binding to a C1q protein. In some embodiments, the Fc region is derived from a human IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the Fc region is incapable of inducing complement activity and / or incapable of inducing antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the Fc region comprises one or more modifications, including, but not limited to, an amino acid substitution. In certain embodiments, the Fc region of a humanized anti-C1q antibody 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 prevents the Fc region from interacting with an Fc receptor. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 is a leucine to glutamate amino acid substitution. 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 a serine to proline amino acid substitution. 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% identity to the amino acid sequence of SEQ ID NO:47.

[0125] Anti-C1q Fab fragment (e.g., FabA) All anti-C1q antibody Fab fragment sequences are incorporated by reference from US Pat. No. 10,723,788, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0126] In certain embodiments, the present disclosure provides a method for the production of a heavy chain (V H / C H 1) and light chain (V L / C L ) an anti-C1q antibody Fab fragment that binds to C1q protein, the anti-C1q antibody Fab fragment comprising six complementarity determining regions (CDRs) (three of which are V L and V H The heavy chain of the antibody Fab fragment is cleaved after the first heavy chain domain of IgG1 (SEQ ID NO: 39) and comprises the following amino acid sequence:

[0127] TIFF2025533847000014.tif27170

[0128] The complementarity determining regions (CDRs) of SEQ ID NO: 39 are shown in bold and underlined text.

[0129] The light chain domain of the antibody Fab fragment comprises the following amino acid sequence (SEQ ID NO: 40):

[0130] TIFF2025533847000015.tif27170

[0131] The complementarity determining regions (CDRs) of SEQ ID NO: 40 are shown in bold and underlined text.

[0132] 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.

[0133] Mab1-Fab is the Fab of the Mab1 (M1) antibody.

[0134] Mab2-Fab is the Fab of the Mab2 antibody.

[0135] Anti-C1q single-arm antibody All anti-C1q single-arm antibody sequences are disclosed in U.S. Patent Application Nos. 63 / 288,883 and 63 / 288,885, which are incorporated herein by reference for the antibodies and related compositions they disclose.

[0136] In certain aspects, the present disclosure provides an antibody that binds to a protein in the complement cascade, such as a C1q protein. 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 and a second Fc polypeptide. The Fc region may comprise an Fcγ receptor binding site mutation. The antibody may be of the IgG4 class. In some embodiments, one of the Fc polypeptides, but not both, 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.

[0137] TIFF2025533847000016.tif24170

[0138] The complementarity determining regions (CDRs) of SEQ ID NO: 40 are shown in bold and underlined text. In some embodiments, the light chain variable domain HVR-L1 has the sequence RASKSINKYLA (SEQ ID NO: 5), the light chain variable domain HVR-L2 has the sequence SGSTLQS (SEQ ID NO: 6), and the light chain variable domain HVR-L3 has the sequence QQHNEYPLT (SEQ ID NO: 7).

[0139] The light chain of the single-arm antibody may comprise the following light chain variable domain amino acid sequence:

[0140] TIFF2025533847000017.tif17170

[0141] The single-arm antibody may comprise a light chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO: 10, 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).

[0142] The single-arm antibody may comprise the following amino acid sequence of a kappa light chain variable domain variant 1 (Vκ1):

[0143] TIFF2025533847000018.tif17170The hypervariable regions (HVRs) of Vκ1 are shown in bold and underlined text.

[0144] The single-arm antibody may comprise the following amino acid sequence of a kappa light chain variable domain variant 2 (Vκ2):

[0145] TIFF2025533847000019.tif17170The hypervariable regions (HVRs) of Vκ2 are shown in bold and underlined text.

[0146] The single-arm antibody may comprise the following amino acid sequence of a kappa light chain variable domain variant 3 (Vκ3):

[0147] TIFF2025533847000020.tif17170The hypervariable regions (HVRs) of Vκ3 are shown in bold and underlined text.

[0148] The single-arm antibody may comprise the following amino acid sequence of a kappa light chain variable domain variant 4 (Vκ4):

[0149] TIFF2025533847000021.tif17170The hypervariable regions (HVRs) of Vκ4 are shown in bold and underlined text.

[0150] The single-arm antibody may comprise a light chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NOs: 11-14, while retaining HVR-L1 RASKSINKYLA (SEQ ID NO: 5), HVR-L2 SGSTLQS (SEQ ID NO: 6), and HVR-L3 QQHNEYPLT (SEQ ID NO: 7).

[0151] The antibody may be of the IgG4 class. The sequence of the IgG4 heavy chain is:

[0152] TIFF2025533847000022.tif70170

[0153] The domains of IgG4 are as follows: CH1: 1-98, hinge: 99-110, CH2: 111-220, and CH3: 221-327. The IgG4 may contain mutations, such as an S108P mutation (for IgG4 arm exchange), an L115E mutation (for FcR binding), a T246W mutation (for knob-in-hole mutation), a T246S mutation (for knob-in-hole mutation), an L248A mutation (for knob-in-hole mutation), a Y187V mutation (for knob-in-hole mutation), and / or an N187A (aglycosylated for FcR binding), an N187Q (aglycosylated for FcR binding), or an N187G (aglycosylated for FcR binding).

[0154] One heavy chain (heavy chain 1 domain) of the single-arm antibody may comprise the following amino acid sequence (SEQ ID NO:2):

[0155] TIFF2025533847000023.tif49170

[0156] The complementarity determining regions (CDRs) of SEQ ID NO: 2 are shown in bold and underlined text. The knob-in-hole T366W mutation of SEQ ID NO: 2 (corresponding to the IgG4 T246W mutation) is shown in underlined text. The S241P (corresponding to the S108P in the case of IgG4 arm swapping) and L248E (corresponding to the L115E mutation in the case of FCR) mutations are shown in bold. In some embodiments, HVR-H1 of the heavy chain variable domain has the sequence GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 of the heavy chain variable domain has the sequence VIHPNSGSINYNEKFES (SEQ ID NO: 10), and HVR-H3 of the heavy chain variable domain has the sequence ERDSTEVLPMDY (SEQ ID NO: 11).

[0157] One heavy chain (heavy chain 1 domain) of the single-arm antibody may comprise the following amino acid sequence (SEQ ID NO: 20):

[0158] TIFF2025533847000024.tif49170

[0159] The complementarity determining regions (CDRs) of SEQ ID NO: 20 are shown in bold and underlined text. The knob-in-hole T366W mutation of SEQ ID NO: 20 (corresponding to the IgG4 T246W mutation) is shown in underlined text. The S241P (corresponding to S108P in the case of IgG4 arm swapping) and L248 (corresponding to the L115E mutation) mutations are shown in bold.

[0160] The antibody may be of the IgG1 class. The sequence of the IgG1 heavy chain is:

[0161] TIFF2025533847000025.tif70170

[0162] The domains of IgG1 are as follows: CH1: 1-98, hinge: 99-110, CH2: 111-223, and CH3: 224-330. IgG1 may contain mutations, such as an L117A mutation (for FcR binding), an L118A mutation (for FcR binding), a T249W mutation (for knob-in-hole mutation), a T249S mutation (for knob-in-hole mutation), an L251A mutation (for knob-in-hole mutation), and / or a Y290V mutation (for knob-in-hole mutation).

[0163] One heavy chain (heavy chain 1 domain) of the single-arm antibody may comprise the following amino acid sequence (SEQ ID NO:21):

[0164] TIFF2025533847000026.tif49170

[0165] The complementarity determining regions (CDRs) of SEQ ID NO: 21 are shown in bold and underlined text. The knob-in-hole T366W mutation of SEQ ID NO: 21 (corresponding to the IgG1 T249W mutation) is shown in underlined text. The L234A (corresponding to the IgG1 L117A mutation) and L235A (corresponding to the IgG1 L1178 mutation) mutations are shown in bold text.

[0166] Heavy chain 1 of the single-arm antibody can comprise the following heavy chain variable domain amino acid sequence:

[0167] TIFF2025533847000027.tif22170

[0168] The single-arm antibody may comprise a heavy chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO: 15, preferably while retaining HVR-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO: 10), and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0169] The single-arm antibody may comprise the following amino acid sequence of heavy chain variable domain variant 1 (VH1):

[0170] TIFF2025533847000028.tif22170The hypervariable regions (HVRs) of VH1 are shown in bold and underlined text.

[0171] The single-arm antibody may comprise the following amino acid sequence of the heavy chain variable domain variant 2 (VH2):

[0172] TIFF2025533847000029.tif22170The hypervariable regions (HVRs) of VH2 are shown in bold and underlined text.

[0173] The single-arm antibody may comprise the following amino acid sequence of the heavy chain variable domain variant 3 (VH3):

[0174] TIFF2025533847000030.tif22170The hypervariable regions (HVRs) of VH3 are shown in bold and underlined text.

[0175] The single-arm antibody may comprise the following amino acid sequence of heavy chain variable domain variant 4 (VH4):

[0176] TIFF2025533847000031.tif22170The hypervariable regions (HVRs) of VH4 are shown in bold and underlined text.

[0177] The single-arm antibody may comprise a heavy chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NOs: 16-19, while retaining HVR-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO: 10), and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0178] The second heavy chain (heavy chain 2 domain) of the N-terminally truncated single-arm antibody may comprise the following amino acid sequence (SEQ ID NO:3):

[0179] TIFF2025533847000032.tif27170

[0180] The heavy chain variable domain and CDRs are absent from SEQ ID NO: 3. The knob-in-hole T366S / L368A / Y407V mutations in SEQ ID NO: 3 are shown in underlined text. The S241P and L248E mutations are shown in bold.

[0181] The second heavy chain (heavy chain 2 domain) of the N-terminally truncated single-arm antibody may comprise the following amino acid sequence (SEQ ID NO: 42):

[0182] TIFF2025533847000033.tif27170

[0183] The second heavy chain (heavy chain 2 domain) of the N-terminally truncated single-arm antibody may comprise the following amino acid sequence (SEQ ID NO: 43):

[0184] TIFF2025533847000034.tif27170

[0185] The heavy chain variable domain and CDRs are absent from SEQ ID NO: 43. The knob-in-hole T366S / L368A / Y407V mutations in SEQ ID NO: 43 are shown in underlined text. The L234A and L235A mutations are shown in bold.

[0186] The second heavy chain (heavy chain 2 domain) of the antibody can comprise any one of the following amino acid sequences (SEQ ID NOs: 44-47):

[0187] TIFF2025533847000035.tif49170

[0188] TIFF2025533847000036.tif49170

[0189] TIFF2025533847000037.tif49170

[0190] TIFF2025533847000038.tif49170

[0191] The second heavy chain (heavy chain 2 domain) of the antibody can comprise any one of the following amino acid sequences (SEQ ID NOS: 48-51):

[0192] TIFF2025533847000039.tif49170

[0193] TIFF2025533847000040.tif49170

[0194] TIFF2025533847000041.tif49170

[0195] TIFF2025533847000042.tif49170

[0196] The CDRs in the second heavy chain variable domain are mutated in SEQ ID NOs: 44-51 to prevent binding to C1q. The CDR mutations are shown in bold and underlined text. The knob-in-hole T366S / L368A / Y407V mutations in SEQ ID NOs: 44-51 are shown in underlined text.

[0197] In some embodiments, the antibody that binds C1q is a light chain domain comprising the amino acid sequence of SEQ ID NO: 40; a first heavy chain domain comprising the amino acid sequence of SEQ ID NO: 2; a second heavy chain domain comprising the amino acid sequence of SEQ ID NO: 3, wherein the second heavy chain domain is an N-terminally truncated heavy chain.

[0198] Nucleic acids, vectors, and host cells 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. Patent No. 4,816,567. In some embodiments, isolated nucleic acids having a nucleotide sequence encoding any of the antibodies of the present disclosure are provided. Such nucleic acids include those encoding the V of anti-C1q antibodies. L / C L and / or V H / C H In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. Host cells containing such nucleic acids may also be provided. The host cells may encode an amino acid sequence comprising (1) the V of the antibody. L / C L an amino acid sequence containing H / C H (2) a vector containing a nucleic acid encoding an amino acid sequence containing 1, or (3) a V L / C L a first vector comprising a nucleic acid encoding an amino acid sequence comprising H / C H The host cell may comprise (e.g., be transduced with) a second vector comprising a nucleic acid encoding an amino acid sequence comprising 1. In some embodiments, the host cell is eukaryotic, 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 E. coli.

[0199] Disclosed herein are methods for producing 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 expression of the antibody. In some embodiments, the antibody is then recovered from the host cell (or host cell culture medium).

[0200] For recombinant production of a humanized anti-C1q antibody of the present disclosure, nucleic acid encoding the antibody is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains).

[0201] Suitable vectors containing a nucleic acid sequence encoding an antibody of the present disclosure, or any of the fragment polypeptides thereof (including antibodies) described herein, include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques or selected from a wide variety of cloning vectors available in the art. While the cloning vector selected can vary depending on the host cell intended for use, useful cloning vectors generally possess the ability to autonomously replicate, possess a unique target for a particular restriction endonuclease, and / or possess a gene for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, e.g., pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Stratagene, and Invitrogen.

[0202] A vector containing a nucleic acid of interest can be introduced into a host cell by any of a number of suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other agents; particle bombardment; lipofection; and infection (e.g., the vector is an infectious agent such as vaccinia virus). The choice of introducing a vector or polynucleotide often depends on the characteristics of the host cell. In some embodiments, the vector contains a nucleic acid containing one or more amino acid sequences encoding an anti-C1q antibody of the present disclosure.

[0203] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells. For example, the anti-C1q antibodies of the present disclosure can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. Expression of antibody fragments and polypeptides in bacteria is described, for example, in U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; and Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254 (describing the expression of antibody fragments in E. coli). In other embodiments, antibodies of the present disclosure can be produced in eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphoid cells (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 antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0204] Pharmaceutical Compositions, Formulations and Administration The present disclosure is generally directed to pharmaceutical compositions comprising the anti-C1q antibodies disclosed herein, including antibody Fab fragments and antibody derivatives. As used herein, the phrase "pharmaceutical composition (also referred to as "pharmaceutical formulation")" refers to a combination of at least one active ingredient (e.g., the anti-C1q antibodies disclosed herein, including antibody Fab fragments and antibody derivatives) and at least one inactive ingredient that, when combined with the active ingredient and / or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal.

[0205] Formulations of anti-C1q antibodies (including antibody Fab fragments and antibody derivatives) have been identified. Such formulations are essentially isotonic and have been formulated at high concentrations (e.g., greater than about 75 mg / ml). In certain embodiments, the formulations have demonstrated high levels of stability. Such formulations have been shown to be compliant with the Subvisible Particulate Matter USP for Ophthalmic Products. <789> Examples of selected formulations include 5-25 mM sodium citrate or sodium succinate (e.g., 5 mM, 10 mM, 15 mM, 20 mM, 25 mM sodium citrate or sodium succinate), 3-7% trehalose (e.g., 3%, 4%, 5%, 6%, 7% (w / v) trehalose), 0.04%-0.06% polysorbate 20 or polysorbate 80 (e.g., 0.04%, 0.05%, 0.06% (w / v) ) polysorbate 20 (PS20) or polysorbate 80 (PS80)), and 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 at pH 6.4 to 7.5 (e.g., 6.4, 6.6, 6.8, 7.0, 7.2, 7.4). The pharmaceutical composition may further comprise 30 mM to 50 mM NaCl (e.g., 30 mM, 40 mM, 50 mM NaCl).

[0206] 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.

[0207] The pharmaceutical composition may further comprise a stabilizer, such as trehalose, to reduce osmotic pressure and maintain the composition in an isotonic state. The trehalose may be about 3% to about 7% (w / v). In some embodiments, the trehalose may be 3%, 4%, 5%, 6%, or 7% (w / v). In some embodiments, the trehalose may be 5% (w / v).

[0208] The pharmaceutical composition may further include a surfactant such as PS20 or PS80 to minimize aggregation (reduction of subvisible particulates). PS20 or PS80 may be 0.04% to 0.06%. In some embodiments, PS20 may be 0.06% (w / v). In some embodiments, PS80 may be 0.06% (w / v).

[0209] The pharmaceutical composition may have a pH of 6.4 to 7.5, hi some embodiments, the pharmaceutical composition may have a pH of about 7.2.

[0210] 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.

[0211] In certain aspects, provided herein is a pharmaceutical composition comprising: (a) at least 75 mg / ml of anti-C1q antibody; 5-25 mM sodium citrate or sodium succinate; (c) 3%-7% trehalose; and (d) 0.04%-0.06% polysorbate 20 or polysorbate 80, wherein the pharmaceutical composition is at a pH of 6.4-7.5.

[0212] In certain embodiments, pharmaceutical compositions contain 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. Pharmaceutical compositions may contain 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, pharmaceutical compositions may contain about 75 mg / ml, 100 mg / ml, 125 mg / ml, 150 mg / ml, 175 mg / ml, 200 mg / ml, 225 mg / ml, or 250 mg / ml of anti-C1q antibody Fab fragments. The pharmaceutical composition may contain 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.

[0213] In certain embodiments, 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, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM sodium citrate or sodium succinate. The sodium citrate may be 10 mM. The sodium succinate may be 5 mM to 20 mM. The pharmaceutical composition may comprise 5 mM to 10 mM, 10 mM to 15 mM, 15 mM to 20 mM, or 20 mM to 25 mM sodium citrate or sodium succinate.

[0214] In certain embodiments, the pharmaceutical composition contains about 3%, 4%, 5%, 6%, or 7% of a stabilizer. The pharmaceutical composition may contain 3% to 4%, 4% to 5%, 5% to 6%, or 6% to 7% trehalose. Stabilizers such as trehalose increase osmotic pressure. Trehalose may be present in an amount of about 4% to about 6%, preferably about 5% (w / v).

[0215] In certain embodiments, the pharmaceutical composition contains about 0.04%, 0.05%, or 0.06% of a surfactant to minimize aggregation (reduction of subvisible particulates). The surfactant may be polysorbate 20 (PS20) or polysorbate 80 (PS80). The pharmaceutical composition may contain 0.04% to 0.05% or 0.05% to 0.06% of polysorbate 20 or polysorbate 80.

[0216] In certain embodiments, the pharmaceutical composition is at a pH of 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 pharmaceutical composition can be at a pH of 6.0-6.5, 6.5-7.0, or 7.0-7.5.

[0217] Optionally, the pharmaceutical composition further comprises 30 mM to 50 mM NaCl. The pharmaceutical composition may comprise about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM NaCl. NaCl may be added for isotonicity. The pharmaceutical composition may comprise 30 mM to 35 mM, 35 mM to 40 mM, 40 mM to 45 mM, or 45 mM to 50 mM NaCl. The amount of NaCl may depend on the amount of trehalose in the formulation. The more trehalose added, the less NaCl is required. The amount of NaCl may also depend on the antibody concentration.

[0218] In certain embodiments, the viscosity of the pharmaceutical composition is 10 cP or less at 25° C. The viscosity of the pharmaceutical composition can 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 can be 1 cP to 5 cP or 5 cP to 10 cP at 25° C.

[0219] The pharmaceutical composition may 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 may be stable at a temperature of 25°C or higher. In some embodiments, the pharmaceutical composition may be stable at a temperature of about 25°C to about 40°C. The pharmaceutical composition may be stable at a temperature of about 25°C, about 30°C, about 35°C, or about 40°C.

[0220] In some embodiments, the pharmaceutical composition is stable for at least 1 year, at least 1.5 years, or at least 2 years. The pharmaceutical composition may be stable at temperatures between -20°C and 25°C. The pharmaceutical composition may be stable at temperatures 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 temperatures between 2°C and 8°C. The pharmaceutical composition may be stable at temperatures above 25°C, for example, about 25°C to about 40°C, preferably about 25°C, about 30°C, about 35°C, or about 40°C.

[0221] The pharmaceutical composition may 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 may be stable after at least one day, at least two days, or at least three days of continuous agitation.

[0222] 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 the syringe. In some embodiments, the syringe has a fill volume of 300 microliters or less. In some embodiments, the syringe delivers a volume of 25 to 100 microliters.

[0223] In certain embodiments, the pharmaceutical composition is prepared for subcutaneous injection. The pharmaceutical composition can be contained in a syringe, an auto-injector, or a pen. In some embodiments, the syringe, the auto-injector, or the pen has a fill volume of 10 ml or less.

[0224] In certain embodiments, the pharmaceutical composition is prepared for intravenous injection. The pharmaceutical composition may be contained in an intravenous solution bag.

[0225] In certain embodiments, the pharmaceutical compositions provided herein exhibit a high level of stability. As used herein with respect to a pharmaceutical composition, the term "stable" means that the antibody within the pharmaceutical composition retains an acceptable degree of structure and / or function and / or biological activity after storage for a defined period of time. A composition may be stable even if the antibody contained therein does not maintain 100% of its structure and / or function and / or biological activity after storage for a defined period of time. Under certain circumstances, retention of about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure and / or function and / or biological activity of the antibody after storage for a defined period of time may be considered "stable."

[0226] Stability can be measured, inter alia, by determining the percentage of native antibody remaining in a composition after storage for a defined period of time at a given temperature. A "native antibody" refers to an antibody having a "native conformation," which refers to the secondary, tertiary, and / or quaternary structure of the antibody in its biologically active state. For example, a native antibody is in monomeric form. Several methods based on physical parameters are available for analyzing and quantifying aggregated antibodies and determining the amount of aggregated and monomeric antibodies. The percentage of native or monomeric antibody can be determined, inter alia, by size exclusion chromatography (e.g., size exclusion high-performance liquid chromatography [SE-HPLC]). As used herein, "acceptable degree of stability" means that at least 90% of the native or monomeric form of the antibody can be detected in the composition after storage for a defined period of time at a given temperature. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the native or monomeric form of the antibody can be detected in the composition after storage at a given temperature for a defined period of time. The defined period after which 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 more. The pharmaceutical composition may 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 may be stable for at least 2 years.When assessing stability, the pharmaceutical composition may be stored at any temperature between about −20° C. and 25° C., for example, 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 between 2° C. and 8° C. The pharmaceutical composition may be stable at temperatures above 25° C., for example, about 25° C. to about 40° C., preferably about 25° C., about 30° C., about 35° C., or about 40° C. For example, a pharmaceutical composition may be considered stable if greater than about 90%, 95%, 96%, or 97% of the native or monomeric antibody is detected by SE-HPLC after 3 months of storage at 5° C. A pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the native or monomeric antibody is detected by SE-HPLC after 6 months of storage at 5° C. A pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the native or monomeric antibody is detected by SE-HPLC after 9 months of storage at 5° C. A pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the native or monomeric antibody is detected by SE-HPLC after 3 months of storage at 25° C. A pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the native or monomeric antibody is detected by SE-HPLC after 6 months of storage at 25° C. A pharmaceutical composition may also be considered stable if greater than about 90%, 95%, 96%, or 97% of the native or monomeric antibody is detected by SE-HPLC after 9 months of storage at 25° C.

[0227] Other methods can be used to assess the stability of the pharmaceutical compositions disclosed herein, such as SEC-HPLC with HIAC, visible particulate testing, and light obscuration for measuring aggregation, visible particulates, and subvisible particulates, respectively. Another stability-indicating method is cation exchange HPLC, which has also been used to detect changes in charge variant profile for stability assessment.

[0228] Stability can also be assessed by measuring the biological activity and / or binding affinity of the antibody to its target. For example, a composition disclosed herein can be considered stable if, after storage for a defined period of time (e.g., 1-12 months) at, e.g., 5°C, 25°C, 40°C, etc., the anti-C1q antibody contained within 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 within the composition prior to storage. Additional methods for assessing the stability of an antibody in a composition are demonstrated in the Examples provided below.

[0229] The stability of a pharmaceutical composition can be evaluated by following the evolution of its physical and chemical parameters over time. Physical stability includes visual and olfactory data, as well as tracking of invisible particulates. To study chemical stability, analytical methods are used to achieve the separation and even quantification of any degradation products. Monitoring the concentration of the active ingredient and the appearance of degradation products over time is completed using methods known to those skilled in the art, including, for example, UV280 (for concentration), and SEC-HPLC, sodium dodecyl sulfate capillary gel electrophoresis (CE-SDS), and cation exchange HPLC (for the appearance of degradation products). Pharmaceutical compositions are prepared according to USP <789> , U.S.P. <787> , or USP <788> It can be stable if it complies with the constraints

[0230] As used herein, with respect to the pharmaceutical compositions disclosed herein, a low level of viscosity would refer to an absolute viscosity of less than about 20 cPoise (cP) at 25° C. For example, a pharmaceutical composition disclosed herein would be considered to have a "low viscosity" 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., as measured using standard viscosity measurement techniques. As used herein, a moderate level of viscosity would refer to an absolute viscosity of about 30 cP to about 20 cP at 25° C. For example, a pharmaceutical composition disclosed herein would be considered to have a "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.

[0231] In some embodiments, the osmolality of the pharmaceutical composition is within the physiological osmolality range of 250-400 mOsm / kg. In some embodiments, the viscosity of the pharmaceutical composition is 30 cP or less at 25° C. In some embodiments, the viscosity of the pharmaceutical composition is 15 cP or less at 25° C. In some embodiments, the viscosity of the pharmaceutical composition is about 10 cP, about 11 cP, about 12 cP, about 13 cP, about 14 cP, about 15 cP, about 16 cP, about 17 cP, about 18 cP, about 19 cP, about 20 cP, about 21 cP, about 22 cP, about 23 cP, about 24 cP, about 25 cP, about 26 cP, about 27 cP, about 28 cP, about 29 cP, or about 30 cP at 25° C.

[0232] Administration Disclosed herein are methods for administering a pharmaceutical composition comprising at least 75 mg / ml of an anti-C1q antibody. The pharmaceutical composition may further comprise 5 to 25 mM sodium citrate or sodium succinate. It may also comprise 3% to 7% trehalose; and / or 0.04% to 0.06% polysorbate 20 or polysorbate 80. The pharmaceutical composition may have a pH of 6.4 to 7.5. The anti-C1q antibody may comprise a light chain variable domain and a heavy chain variable domain. The antibody may bind to 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 antibody light chain variable domain, antibody fragment thereof, and / or antibody derivative thereof comprises HVR-L1, HVR-L2, and HVR-L3 of monoclonal antibody M1 produced by the hybridoma cell line deposited under ATCC Accession No. PTA-120399. The antibody heavy chain variable domain, antibody fragment thereof, and / or antibody derivative thereof comprises HVR-H1, HVR-H2, and HVR-H3 of monoclonal antibody M1 produced by the hybridoma cell line deposited under ATCC Accession No. PTA-120399.

[0233] 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 for HVR-L1, SEQ ID NO: 6 for HVR-L2, SEQ ID NO: 7 for HVR-L3, SEQ ID NO: 9 for HVR-H1, SEQ ID NO: 10 for HVR-H2, and SEQ ID NO: 11 for HVR-H3.

[0234] The antibody may comprise a light chain variable domain amino acid sequence at least 85%, 90%, or 95% identical to SEQ ID NO:4, preferably 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 at least 85%, 90%, or 95% identical to SEQ ID NO:8, preferably retaining HVR-H1 GYHFTSYWMH (SEQ ID NO:9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO:10), and HVR-H3 ERDSTEVLPMDY (SEQ ID NO:11).

[0235] 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 suborbital, intravitreal, intraocular, subconjunctival, retrobulbar, peribulbar, and / or intrathecal administration. In certain embodiments, the pharmaceutical composition is administered by suborbital, intravitreal, intraocular, subconjunctival, retrobulbar, peribulbar, and / or intrathecal injection.

[0236] In certain embodiments, the pharmaceutical composition is administered in a delivery volume of 3 ml, 2.5 ml, 2 ml, 1.5 ml, or 1 ml or less. In some embodiments, the pharmaceutical composition is administered in a delivery volume of 2 ml or less. In some embodiments, the pharmaceutical composition is administered in a delivery volume of 300 microliters or less. In some embodiments, the pharmaceutical composition is administered in a delivery volume of 25 microliters, 50 microliters, 75 microliters, or 100 microliters or less.

[0237] In certain aspects, the present disclosure relates to a syringe containing the pharmaceutical composition disclosed herein. The syringe can be for subcutaneous, intravitreal, or intraocular injection. In some embodiments, the syringe has a delivery volume of 10 μl or less. In some embodiments, the syringe has a needle size of 25 G (25 gauge) or less. 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 still other embodiments, the syringe is an automatic disposable fixed dose autoinjector. In some embodiments, the syringe has a delivery volume of 300 microliters or less. The syringe can be a syringe.

[0238] In certain aspects, the present disclosure relates to a pre-filled syringe containing the pharmaceutical composition disclosed herein. The pre-filled syringe can contain a delivery volume of 300 microliters or less.

[0239] In some embodiments, the pharmaceutical compositions provided herein can be contained in any container suitable for storing pharmaceuticals and other therapeutic compositions. For example, the pharmaceutical compositions can be contained in a sealed, sterile plastic or glass container having a defined volume, such as a vial, ampoule, syringe, cartridge, or bottle. For example, different types of vials, including clear and opaque (e.g., amber) glass or plastic vials, can be used to contain the compositions provided herein. Similarly, any type of syringe can be used to contain and / or administer the pharmaceutical compositions disclosed herein.

[0240] The pharmaceutical compositions provided herein are silicone-free and have low particulate levels, i.e., meet USP standards for subvisible particulate matter. <789> (USP) limits (less than 50 particles per ml for 10 μm particles, less than 5 particles per ml for 25 μm particles). <789> (See USP 35-NF 30. MD, USA: The United States Pharmacopeial Convention; 2012). Particulate matter in ophthalmic solutions. USP 35-NF 30. MD, USA: The United States Pharmacopeial Convention; 2012). For example, Terumo's i-coated stopper technology eliminates the need for silicone oil in syringe systems while providing consistent and predictable break-loose and glide forces. Eliminating the use of silicone oil in pre-fill syringe (PFS) systems significantly reduces the subvisible particulate load, allowing for the reduction of subvisible particulate matter in ophthalmic solutions. <789> Allows you to meet stringent particulate requirements such as fine particulate matter.

[0241] The pharmaceutical compositions provided herein may be contained in "normal tungsten" syringes or "low tungsten" syringes. As will be understood by those skilled in the art, the process of making glass syringes generally involves the use of a hot tungsten rod, which functions to puncture the glass, thereby creating a hole through which liquid can be drawn and expelled from the syringe. This process results in the deposition of trace amounts of tungsten on the interior surface of the syringe. Subsequent cleaning 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 can contain less than about 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or even fewer ppb of tungsten.

[0242] Rubber plungers used in syringes and rubber stoppers used to close the openings of vials can be coated to prevent contamination of the pharmaceutical contents of the syringe or vial and / or to maintain their stability. Thus, according to certain embodiments, the pharmaceutical compositions provided herein can be contained in a syringe with 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 use with vials and syringes containing the pharmaceutical compositions disclosed herein are found in, for example, U.S. Patent 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 entireties. Certain exemplary coated rubber stoppers and plungers that may be used in the methods disclosed herein are commercially available under the trade name "FluroTec®" and are available from West Pharmaceutical Services, Inc. (Lionville, Pa.).

[0243] In certain embodiments, the pharmaceutical composition may be administered to the patient by intravitreal or intraocular injection.

[0244] In certain embodiments, the pharmaceutical compositions may be administered to a patient by a parenteral route, such as by injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, suborbital, intravitreal, intraocular, subconjunctival, retrobulbar, peribulbar, and / or intrathecal injection). A number of reusable pen and / or autoinjector delivery devices may be used to subcutaneously deliver the pharmaceutical compositions disclosed herein. Examples include AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.), NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN™, to name a few. Examples of disposable pen and / or auto-injector delivery devices applicable for subcutaneous delivery of the pharmaceutical compositions disclosed herein include, but are not limited to, the SOLOSTAR pen (sanofi-aventis), FLEXPEN (Novo Nordisk), and KWIKPEN (Eli Lilly), the SURECLICK auto-injector (Amgen, Thousand Oaks, Calif.), PENLET (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA Pen (Abbott Labs, Abbott Park, Ill.), to name a few.

[0245] The use of a microinfuser to deliver the pharmaceutical compositions disclosed herein is also contemplated herein. As used herein, the term "microinfuser" refers to a subcutaneous delivery device designed to slowly administer large volumes (e.g., up to about 2.5 mL or more) of a therapeutic composition over an extended period of time (e.g., about 10, 15, 20, 25, 30 minutes or more). See, for example, U.S. Patent Nos. 6,629,949, 6,659,982, and Meehan et al., J. Controlled Release 46:107-116 (1996).

[0246] Treatment method In certain aspects, the present disclosure relates to methods of preventing, reducing the risk of developing, or treating a disease or disorder associated with the complement pathway, comprising administering a pharmaceutical composition disclosed herein. The present disclosure is also generally directed to methods of preventing, reducing the risk of developing, or treating a disease or disorder associated with the complement pathway, comprising administering a pharmaceutical composition using a syringe, injector, or intravenous solution bag disclosed herein.

[0247] The present invention provides a method for modulating immune responses to diseases disclosed herein through the administration of agents that are complement antagonists. For example, without being bound by theory, immature astrocytes induce the expression of C1q protein in neurons during development. Inflammatory mediators, such as complement factors, are normally expressed at very low levels in healthy brain tissue but can be rapidly induced by various insults to the brain, such as infection, ischemia, and injury. Activation of C1q, C1r, and C1s contributes to the inflammatory response that leads to synaptic loss, as well as the occurrence and recurrence of seizures and seizure-related neuronal damage. During the developmental process of neurodegenerative diseases, overexpression of C1q, C1r, and C1s can be linked to signals for complement activation, such as cytokines such as β-amyloid, APP, IFNγ, and TNFα, leading to inflammation.

[0248] By administering the pharmaceutical compositions disclosed herein that inhibit complement activation, synapses that would otherwise be lost can be preserved.

[0249] The method neutralizes a biological activity of complement. The affected biological activity of complement can be (1) C1q binding to autoantibodies, (2) C1q binding to C1r, (3) C1q binding to C1s, (4) C1q binding to IgM, (5) C1q binding to phosphatidylserine, (6) C1q binding to pentraxin-3, (7) C1q binding to C-reactive protein (CRP), (8) C1q binding to globular C1q receptor (gC1qR), (9) C1q binding to complement receptor 1 (CR1), (10) C1q binding to beta-amyloid, (11) C1q binding to calreticulin, (12) C1q binding to apoptotic cells, or (13) C1q binding to B cells. The affected complement biological activities may further include (1) activation of the classical complement pathway, (2) activation of antibody and complement-dependent cytotoxicity, (3) CH50 hemolysis, (4) synapse loss, (5) B cell antibody production, (6) dendritic cell maturation, (7) T cell proliferation, (8) cytokine production, (9) microglial activation, (10) Arthus reaction, (11) phagocytosis of synapses or nerve terminals, or (12) activation of complement receptor 3 (CR3 / C3)-expressing cells.

[0250] The method promotes the improved maintenance of neuronal function in pathologies associated with synapse loss.Maintaining neural connections provides functional improvement in neurodegenerative diseases compared to untreated patients.Preventing synapse loss can include at least measurable improvement, such as at least 10% improvement, at least 20% improvement, at least 50% improvement, or more in synapse number compared to controls that do not undergo such treatment for 1, 2, 3, 4, 5, 6 days or at least 1 week.

[0251] In one aspect, described herein is a method for 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 as a result of a neurodegenerative disorder, a central nervous system disorder, or a peripheral nervous system disorder. In some embodiments, the method further comprises administering neural progenitor cells or a neurogenesis enhancer. In some embodiments, the antibody binds to C1q and inhibits complement activation.

[0252] In one aspect, described herein is a method for treating or preventing a disease associated with complement activation in an individual in need thereof, 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 synapse loss or loss of neural connections, synapse loss dependent on complement receptor 3 (CR3) / C3 or complement receptor CR1, pathological activity-dependent synapse pruning, or synapse phagocytosis by microglia. The neurodegenerative disorder can be Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis, ophthalmic disorders, glaucoma, myotonic dystrophy, Guillain-Barré syndrome (GBS), myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down's syndrome, Parkinson's disease, or Huntington's disease (HD). In some embodiments, the neurodegenerative disorder is ALS, GBS, or HD.

[0253] 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), remote tissue injury after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, glaucoma, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degenerative diseases, age-related macular degeneration (AMD), geographic atrophy, choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, intraocular neovascular disease, diabetic macular edema, pathologic myopia, von Hippel-Lindau disease, ocular histoplasmosis, neuromyelitis optica (NMO), central retinal vein occlusion (CRVO), corneal neovascularization, retinal neovascularization, Leber's hereditary optic neuropathy, optic neuritis, Behçet's retinopathy, ischemic optic neuropathy, retinal vasculitis, ANCA vasculitis, Purcher's retinopathy, Sjogren's dry eye disease, dry AMD, wet AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, multiple sclerosis, allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome (COPD), asthma, aspiration pneumonia, multiple sclerosis, Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis.

[0254] In some embodiments, the disease associated with complement activation is an autoimmune disease selected from myasthenia gravis, type 1 diabetes, Hashimoto's thyroiditis, Addison's disease, celiac disease, Crohn's disease, pernicious anemia, pemphigus vulgaris, vitiligo, autoimmune hemolytic anemia, paraneoplastic 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.

[0255] In some embodiments, the disease associated with complement activation is 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 alloimmunization, Felty 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 syndrome (APS), infection, or a drug-induced hematological disorder.

[0256] As used herein, "neurological" or "cognitive" function refers to the reduction of synapses in the brain to enhance a patient's ability to think, function, etc. As used herein, the term "subject" encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates, e.g., chimpanzees, and other ape and monkey species; farm animals, e.g., cows, horses, sheep, goats, pigs; domestic animals, e.g., rabbits, dogs, and cats; and laboratory animals, including rodents, e.g., rats, mice, and guinea pigs. The term does not denote a particular age or sex.

[0257] The complement inhibitors of the present disclosure may be used in conjunction with any additional treatment, such as immunosuppressive therapy, for any of the diseases disclosed herein, including, but not limited to, autoimmune diseases and / or neurodegenerative diseases.

[0258] In some embodiments, the pharmaceutical compositions disclosed herein are administered in combination with inhibitors of the alternative pathway of complement activation. Such inhibitors may include, but are not limited to, factor B inhibitory antibodies, factor D inhibitory antibodies, constatin-like peptides that block cleavage of soluble, membrane-bound, tagged, or fusion protein forms of CD59, DAF, CR1, CR2, Crry, or C3, non-peptide C3aR antagonists such as SB290157, cobra venom factor, or non-specific complement inhibitors such as nafamostat mesylate (FUTHAN; FUT-175), aprotinin, K-76 monocarboxylic acid (MX-1), and heparin (see, e.g., T. E. Mollnes & M. Kirschfink, Molecular Immunology 43 (2006) 107-121). In some embodiments, the pharmaceutical compositions disclosed herein are administered in combination with inhibitors of the interaction between an autoantibody and its autoantigen. Such inhibitors can include purified soluble forms of autoantigens, or antigen mimics, such as peptide or RNA-derived mimotopes (including mimotopes of AQP4 antigens). Alternatively, such inhibitors can include blocking agents that recognize autoantigens and prevent autoantibody binding without triggering the classical complement pathway. Such blocking agents can include, for example, autoantigen-binding RNA aptamers or antibodies that lack C1q binding sites in the Fc domain (e.g., Fab fragments or antibodies engineered not to bind C1q). [Example]

[0259] Example 1: Formulation development of anti-C1q Fab fragment (Fab A) The feasibility of a highly concentrated anti-C1q antibody Fab fragment formulation at 200 mg / mL was evaluated 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 particulate (SVP) count data indicate that the presence of a surfactant is necessary to stabilize the anti-C1q antibody Fab fragment at 200 mg / mL. [Table 2]

[0260] Polysorbate 20 (PS20) surfactant was added to all formulation candidates. Sorbitol and 2-hydroxypropyl-β-cyclodextrin (2HPBCD) were evaluated as alternatives to trehalose dihydrate. Additionally, the effects of freeze / thaw (F / T) cycling (freeze at -20°C / thaw at room temperature), agitation stress, and accelerated stability were evaluated. [Table 3]

[0261] All samples were clear with no visible particulates at t=0. [Table 4]

[0262] Samples were tested for USP solubility after five freeze / thaw (F / T) cycles. <789> Furthermore, chemical stability after five F / T cycles remained unchanged for all formulation candidates. [Table 5]

[0263] Agitation demonstrated a more severe stress condition compared to freeze / thaw stress. All samples appeared hazy with visible particulates after 5 days of agitation, and the USP <789> The chemical stability after mixing was unchanged. [Table 6]

[0264] Accelerated stability was checked at 8 weeks at 25° C. All samples except SVP were within specification. [Table 7]

[0265] Accelerated stability was checked at 40°C for 8 weeks. All samples were within specification except for SVP. Other notable, though still acceptable, changes included an increase in the percentage of pre-peak and inter-peak species by cation exchange HPLC (CEX-HPLC) and high molecular weight species (HMW) by SEC-HPLC. [Table 8]

[0266] After freeze / thaw cycles and agitation, all samples failed visual appearance and SVP, but passed all other product specifications. All samples passed product specifications except for SVP after 8 weeks of storage at 25°C and 40°C.

[0267] The surfactant levels in the formulation were optimized and met visual appearance acceptance criteria (i.e., no haze, no visible particulates) after at least three freeze / thaw cycles and after at least four weeks of storage at 25°C.

[0268] Subsequent studies focused on SVP and visual appearance tests, which proved to be the most indicative of stability compared to other tests for measuring changes after multiple freeze-thaw cycles and agitation stress.

[0269] The optimal detergent concentration was determined to be in the range of 0.02% to 0.06%. Furthermore, polysorbate 20 (PS20) and polysorbate 80 (PS80) detergents were compared at a fixed concentration of 0.06%. This study was performed at an anti-C1q antibody Fab fragment concentration of 100 mg / mL. [Table 9] [Table 10]

[0270] 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. [Table 11]

[0271] 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. Decreasing the protein concentration did not result in a corresponding decrease in detergent concentration. [Table 12]

[0272] The effect of pH was evaluated at different pHs using sodium succinate and sodium citrate buffers. [Table 13]

[0273] Sodium succinate (pH 5.5) samples gelled when stored at 2-8°C. However, the gel state was reversible and became liquid at room temperature. Both sodium succinate and sodium citrate (pH 6.4) samples remained liquid at 2-8°C after complete thawing, but passed through this brief but significant gel phase during thawing. However, all samples formulated at pH 7.2 did not exhibit gelation behavior, transient or otherwise. [Table 14]

[0274] A pH-dependent gel phase transition was observed, indicating a tendency for product gelation with decreasing pH. Furthermore, no difference in SVP was observed between sodium succinate and sodium citrate at pH 7.2.

[0275] Anti-C1q antibody Fab fragments were formulated and allowed to settle in 10 mM sodium citrate, 5% (w / v) trehalose dihydrate, 40 mM sodium chloride, 0.06% (w / v) polysorbate 20, pH 7.2. [Table 15-1] [Table 15-2] [Table 16-1] [Table 16-2] [Table 17-1] [Table 17-2]

[0276] Incorporation by Reference Each of the patents, published patent applications, and non-patent literature cited herein is hereby incorporated by reference in its entirety.

[0277] equivalent 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 which equivalents are intended to be encompassed by the following claims.

Claims

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

5.

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

7.

3. The pharmaceutical composition of claim 1 or 2, wherein the antibody comprises a heavy chain variable domain comprising HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the 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 single-arm 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 to 38, and the light chain variable domain comprises HVR-L1 having the amino acid sequence of SEQ ID NO: 5, HVR-L2 having the amino acid sequence of SEQ ID NO: 6, and HVR-L3 having the amino acid sequence of SEQ ID NO:

7.

7. 7. The pharmaceutical composition of claim 6, wherein the light chain variable domain comprises an amino acid sequence selected from 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 to 34, and the heavy chain variable domain comprises HVR-H1 having the amino acid sequence of SEQ ID NO: 9, HVR-H2 having the amino acid sequence of SEQ ID NO: 10, and HVR-H3 having the amino acid sequence of SEQ ID NO:

11.

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

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

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

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

39.

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

40.

14. 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. 14. 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. 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 the anti-C1q antibody Fab fragment.

17. 17. The pharmaceutical composition of any one of claims 1-16, 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, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM sodium citrate or sodium succinate.

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

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

21. 21. The pharmaceutical composition of any one of claims 1-20, wherein the pharmaceutical composition is at a pH of 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. 22. The pharmaceutical composition of any one of claims 1 to 21, further comprising 30 mM to 50 mM NaCl.

23. 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 according to any one of claims 1 to 23, wherein the viscosity of the pharmaceutical composition is 10 cP or less at 25°C.

25. 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. 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. 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 2 years.

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

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

32. 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. 33. The pharmaceutical composition of any one of claims 26 to 32, wherein the pharmaceutical composition is stable after at least 1, at least 2, at least 3, at least 4, or at least 5 freeze / thaw cycles.

34. 34. The pharmaceutical composition of any one of claims 26 to 33, wherein the pharmaceutical composition is stable after continuous stirring for at least 1 day, at least 2 days, or at least 3 days.

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

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

37. 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 within a syringe.

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

40. 40. The pharmaceutical composition of claim 38 or 39, wherein the syringe has a fill volume of 300 microliters or less.

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

42. 42. The pharmaceutical composition of any one of claims 38 to 41, wherein the syringe and / or the pharmaceutical composition is silicone-free and / or has low particulate levels.

43. 43. The pharmaceutical composition of any one of claims 38 to 42, wherein the syringe and / or the pharmaceutical composition has less than 50 microparticles per ml of 10 μm microparticles and / or less than 5 microparticles per ml of 25 μm microparticles.

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

45. The pharmaceutical composition according to any one of claims 1 to 34, wherein the pharmaceutical composition is prepared for subcutaneous injection.

46. 46. ​​The pharmaceutical composition of claim 45, wherein the pharmaceutical composition is contained within a syringe, auto-injector, or pen.

47. 47. The pharmaceutical composition of claim 46, wherein the syringe, auto-injector, or pen has a fill volume of 10 ml or less.

48. The pharmaceutical composition according to any one of claims 1 to 34, wherein the pharmaceutical composition is prepared for intravenous injection.

49. 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. 51. The syringe of claim 50, wherein the syringe comprises a delivery volume of 10 ml or less.

52. 52. The syringe of claim 50 or 51, wherein the syringe is an automatic reusable fixed dose pen.

53. 52. The syringe of claim 50 or 51, wherein the syringe is an automatic reusable variable dose pen.

54. 52. The syringe of claim 50 or 51, wherein the syringe is an automatic disposable fixed dose auto-injector.

55. 51. The syringe of claim 50, wherein the syringe comprises a delivery volume of 300 microliters or less.

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

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

58. 58. The pre-filled syringe of claim 57, wherein the pre-filled syringe comprises a delivery volume of 300 microliters or less.

59. 59. The pre-filled syringe of claim 57 or 58, wherein the pre-filled syringe and / or the pharmaceutical composition is silicone-free and / or has low particulate levels.

60. 60. The pre-filled syringe of any one of claims 57 to 59, wherein the pre-filled syringe and / or the pharmaceutical composition has less than 50 microparticles per ml of 10 μm microparticles and / or less than 5 microparticles per ml of 25 μm microparticles.

61. 61. The prefilled syringe of claim 60, wherein the prefilled syringe and / or the pharmaceutical composition conforms to the limits of USP <789>.

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

63. 63. The method of claim 62, wherein the patient is suffering from synapse loss as a result of a neurodegenerative disorder, a central nervous system disorder, or a peripheral nervous system disorder.

64. 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. 50. A method of treating or preventing a disease associated with complement activation in an individual in need thereof, said method comprising administering a pharmaceutical composition according to any one of claims 1 to 49.

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

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

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

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

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

72. 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-Barré syndrome (GBS), myasthenia gravis, bullous pemphigoid, spinal muscular atrophy, Down's syndrome, Parkinson's disease, traumatic brain injury (TBI), epilepsy, or Huntington's disease (HD).

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

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

75. 67. 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 inflammatory disease, the autoimmune disease, the complement-associated eye disease, or the metabolic disorder is selected from the group consisting of diabetes, obesity, rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), remote tissue injury after ischemia and reperfusion, complement activation during cardiopulmonary bypass surgery, dermatomyositis, pemphigus, lupus nephritis and resulting glomerulonephritis and vasculitis, cardiopulmonary bypass, cardioplegia-induced coronary endothelial dysfunction, type II membranoproliferative glomerulonephritis, IgA nephropathy, acute renal failure, cryoglobulinemia, antiphospholipid syndrome, glaucoma, chronic open-angle glaucoma, acute angle-closure glaucoma, macular degenerative disease, age-related macular degeneration (AMD), geographic atrophy, choroidal neovascularization (CNV), uveitis, diabetic retinopathy, ischemia-related retinopathy, endophthalmitis, intraocular neovascularization, and the like.

76. The method of claim 75, wherein the retinal vascular disease is selected from vascular disease, diabetic macular edema, pathologic myopia, von 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, Purcher's retinopathy, Sjogren's dry eye disease, dry AMD, sarcoidosis, temporal arteritis, polyarteritis nodosa, allograft, hyperacute rejection, hemodialysis, chronic obstructive pulmonary distress syndrome (COPD), asthma, aspiration pneumonia, multiple sclerosis, multifocal motor neuropathy (MMN), Guillain-Barré syndrome, myasthenia gravis, bullous pemphigoid, or myositis.

77. 77. The method of claim 76, wherein the disease associated with complement activation is an autoimmune disease selected from 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, paraneoplastic 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 diseases associated with complement activation include cold agglutinin hemolytic anemia (cold agglutinin disease), cold antibody hemolytic anemia, ABO incompatible 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 alloimmunization, Felty syndrome, and antibody-mediated thrombocytopenia.

77. The method of claim 76, wherein the hematological disorder is selected from 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 syndrome (APS), infection, or a drug-induced hematological disorder.

Images