Compositions and methods for treating eye diseases

Abstract

The present disclosure relates generally to compositions and methods for preventing, reducing the risk of developing, or treating inherited retinal diseases (IRDs) (e.g., retinitis pigmentosa, choroideremia, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis), X-linked RP, and Usher syndrome or retinal detachment.

Description

[Technical field]

[0001] Related Applications This patent application claims priority to U.S. Provisional Patent Application No. 63 / 336,539, filed on April 29, 2022, which is incorporated by reference in its entirety herein. [Background technology]

[0002] Inherited retinal diseases (IRDs) are a group of disorders that can cause severe vision loss or even blindness. Each IRD is caused by at least one gene not functioning properly. IRDs (such as retinitis pigmentosa) affect individuals of all ages, can progress at different rates, and are rare. However, most are progressive, meaning that the symptoms of the disease worsen over time. Current approaches to therapy aim to repair genetic defects, but there are numerous genetic defects, and each defect only affects a small number of patients. The only approved treatment, voretigene neparvovec-rzyl, is indicated for the genetic mutation RPE65, but this gene is present in only 1-2% of IRD patients. Another approach uses a device called a "retinal prosthesis" to convert light into electrical energy to stimulate the retina directly, but this approach does not address the pathophysiology of the disease. Currently, there are no approved non-genetic treatments or therapies for IRDs. Thus, there is a significant unmet need for treatments for patients with IRD.

[0003] Retinal detachment is an eye disorder in which the retina detaches from the underlying layer of supporting tissue. Retinal detachment occurs in approximately 10-12 cases per 100,000 per year. In approximately 50% of cases, the central retina detaches, resulting in macula-off retinal detachment. When the central retina detaches, despite successful reattachment of the retina, visual recovery only reaches approximately 50% of pre-detachment visual acuity. The cause of this limited visual recovery is photoreceptor degeneration. There are no approved treatments or therapies to improve visual function after successful macular detachment retinal detachment surgery. Thus, a significant unmet need exists for treatment for patients with retinal detachment. Summary of the Invention

[0004] The present disclosure generally relates to compositions and methods for preventing, reducing the risk of developing, or treating inherited retinal diseases (IRDs) (e.g., retinitis pigmentosa / rod-cone dystrophy, choroideremia, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis, X-linked RP, Usher syndrome) and / or retinal detachment in a human patient. In some embodiments, an anti-C1q antibody is administered prior to, after, and / or concurrently with retinal detachment surgery. Such methods may restore vision in a human patient and / or improve vision in a human patient.

[0005] Such methods include administering to a patient, via intravitreal injection, about 1 mg to about 10 mg of a composition comprising an anti-C1q antibody, the antibody comprising 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, and 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. In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs:4 and 35-38, the 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 light chain variable domain comprises an amino acid sequence selected from SEQ ID NOs:4 and 35-38. In some embodiments, the antibody comprises a heavy chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 8 and 31-34, the 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. In some embodiments, the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 8 and 31-34. In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 4 and 35 to 38, including 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, and 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, including 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.In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, and a heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 8 and 31-34. The antibody may be a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, an antibody fragment, or an antibody derivative thereof. The antibody fragment may be 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 Fab fragment comprises a heavy chain Fab fragment of SEQ ID NO: 39 and a light chain Fab fragment of SEQ ID NO: 40.

[0006] In some embodiments, the antibody is administered once a week, once every two weeks, once every three weeks, once a month, once every four weeks, once every six weeks, once every eight weeks, once every two months, once every ten weeks, once every twelve weeks, once every three months, or once every four months. In some embodiments, the antibody is administered for at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

[0007] In some embodiments, the composition administered comprises about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, or about 10 mg of anti-C1q antibody. The composition administered may comprise about 1 mg to about 5 mg of anti-C1q antibody. The composition administered may comprise about 1 mg to about 2.5 mg, about 2.5 mg to about 5 mg, about 5 mg to about 7.5 mg, or about 7.5 mg to about 10 mg of anti-C1q antibody. The composition administered may comprise about 5 mg of anti-C1q antibody. The composition administered may comprise about 10 mg of anti-C1q antibody. [Brief description of the drawings]

[0008] [Figure 1A]A shows photoreceptor damage and microgliosis after light exposure. B shows immunofluorescence (IF) images and quantification demonstrating progressive loss of photoreceptor synapses (Basson) and cell bodies (Dapi) after light injury. [Figure 1B] A shows photoreceptor damage and microgliosis after light exposure. B shows immunofluorescence (IF) images and quantification demonstrating progressive loss of photoreceptor synapses (Basson) and cell bodies (Dapi) after light injury. [Figure 1C] C shows photoreceptor damage and microgliosis after light exposure. C shows immunofluorescence (IF) images and quantification showing progressive loss of photoreceptor synapses (Basson) and cell bodies (Dapi) after light injury. [Figure 1D] A shows photoreceptor damage and microgliosis after light exposure. B shows IF images and quantification showing increased microglia / macrophage reactivity (Iba1 and CD68) after light injury. The distribution of phagocytic microglia in the synaptic layer peaked on day 1, when significant synaptic loss was first observed. [Figure 1E] A and B show photoreceptor damage and microgliosis after light exposure. C shows IF images and quantification showing increased microglia / macrophage reactivity (Iba1 and CD68) after light injury. The distribution of phagocytic microglia in the synaptic layer peaked on day 1, when significant synaptic loss was first observed. [Figure 1F] A shows photoreceptor damage and microgliosis after light exposure. B shows IF images and quantification showing increased microglia / macrophage reactivity (Iba1 and CD68) after light injury. The distribution of phagocytic microglia in the synaptic layer peaked on day 1, when significant synaptic loss was first observed. [Figure 2A] Figure 1 shows complement signature and C1q distribution in the retina after light exposure. A shows an ELISA assay demonstrating increased levels of the initiating classical complement components C1q and C1s, as well as the downstream activation product C3d, in retinal lysates after light injury. [Figure 2B]A shows complement signature and C1q distribution in the retina after light exposure. B shows an ELISA assay demonstrating increased levels of the initiating classical complement components C1q and C1s, as well as the downstream activation product C3d, in retinal lysates after light injury. [Figure 2C] Figure 1 shows complement signature and C1q distribution in the retina after light exposure.C shows an ELISA assay demonstrating increased levels of the initiating classical complement components C1q and C1s, as well as the downstream activation product C3d, in retinal lysates after light injury. [Figure 2D] Complement signature and C1q distribution in the retina after light exposure. D, IF showing retinal C1q distribution and its co-localization with microglia / macrophages (Iba1) and synapses (Bassoon). [Figure 2E] (E) Complement signature and C1q distribution in the retina after light exposure. Correlation analysis showing a significant negative correlation between C1q levels in the OPL and photoreceptor synapse density, consistent with a causal relationship. [Diagram 3] Figure 1 shows microglial phagocytosis of synapses after light exposure. A shows IF demonstrating increased C1q levels in the OPL of light-damaged retinas and its proximity to Bassoon+ve synapses (i). B shows high-resolution and 3D surface-rendered images showing microglial phagocytosis of C1q-tagged synapses in light-damaged retinas. C-E show quantitative analysis showing a significant decrease in synapse density (C), an increase in the percentage of C1q-tagged synapses (D), and an increase in microglial phagocytosed C1q-tagged synapses (E) in light-damaged retinas compared to naïve. [Figure 4]Phosphatidylserine (PS) externalization on photoreceptor synapses and in vitro binding to C1q. A shows IF showing PSVue labeling of PS in the OPL of light damaged retina. 3D surface rendered images (i-ii) showing PSVue proximity to Bassoon and C1q show PS externalization on synapses. B shows assay showing C1q binding to PS lipid beads. No binding to control phophatidylcholine (PC) beads. C-D shows assay showing deposition of C1q and C4 on serum exposed PS lipid beads. No deposition was observed on PC beads. E-F shows competition assay showing reduced deposition of C1q and C4 on serum exposed PS lipid beads in the presence of anti-C1q neutralizing antibody. [Diagram 5] Figure 1 shows retinal PK / PD following anti-C1q treatment. A-D show PK / PD data showing measurable drug levels in retinal lysates from animals treated with anti-C1q, as well as a significant reduction in C1q, C1s and C3d levels upon anti-C1q treatment. [Figure 6] Figure 1 shows C1q distribution in human GA retina. A-B show IF demonstrating reduced immunoreactivity of presynaptic marker Vglut1 (A) and increased labeling of C1q in the photoreceptor synaptic layer OPL (B) compared to healthy donors, confirming synaptic loss and C1q accumulation occurring in GA retina. C shows triple immunolabeling of C1q (grey), presynaptic maker Vglut1 and postsynaptic marker (HOMER1) confirming colocalization of C1q with photoreceptor synapses in human GA donor retina. [Figure 7] Human C1q binding assay. Mab2-Fab, FabA and Mab2 were bound to human C1q in a one-sided ELISA. Bound antibody or Fab molecules were detected using enzyme-labeled anti-human Fc or anti-human kappa antibodies followed by enzyme substrate. The antibodies showed comparable binding affinity to human C1q. The EC50 of Mab2-Fab, FabA and Mab2 were 4.4, 2.5 and 4.9ng / mL (range 34-95pM), respectively. [Figure 8]FabA inhibits the classical pathway but not the lectin and alternative complement pathways. FabA and Mab2 were assessed for their ability to inhibit the classical, lectin and alternative complement pathways using an ELISA-based assay kit from Eurodiagnostica (Weislab™). Wells were coated with specific activators of the classical (IgM), lectin (mannan) or alternative (lipopolysaccharide) pathways and activation of all pathways was assessed using a C5b-9 terminal complex detection antibody. A C5 inhibitor antibody was used as a positive control. FabA and Mab2 selectively block the classical pathway with IC50≦0.3 μg / mL, while anti-C5 inhibits all three pathways. [Figure 9] Inhibition of hemolysis of IgM-coated RBCs by human serum. Sheep RBCs pre-sensitized with surface-reactive polyclonal IgM antibodies were co-incubated with human serum (diluted 100x) for 20-30 minutes at 37°C. RBC hemolysis was quantified by measuring hemoglobin release and is expressed as a percentage of the hemolysis caused by untreated control. [Figure 10]Figure 1 shows a reduction in the number of damaged axons in the optic nerve of eyes treated with Mab1-Fab, Mab1, or Mab2. In one eye of each animal, 1 μl of 6 μm polystyrene beads, 1 μl of 10 μm polystyrene beads (Polybead Microspheres; Polysciences, Inc., Warrington, PA, USA), and 1 μl of viscoelastic solution (10 mg / mL sodium hyaluronate; Advanced Medical Optics Inc., USA) were injected into the anterior chamber on day 1 to induce an elevation in IOP. The contralateral eye was left untouched to serve as a control. Antibodies Mab2, Mab1, and Mab1-Fab (Fab derived from enzymatic digestion of Mab1), versus saline, were administered to eyes that had received intravitreal microbead injections 1 day before and 1 week after microbead injection (days 0 and 7, 2 μL of 10 mg / mL antibody saline for each injection, versus saline alone). Two weeks after injury, optic nerves were harvested from animals (perfused with saline and 4% paraformaldehyde), post-fixed with 4% paraformaldehyde and 1% osmium, dehydrated in ascending alcohol concentrations, and mounted in 1% uranyl acetate / ethanol. Nerves were embedded in epoxy resin and semi-thin sections (1 um) were cut. The total number of degenerated axons was estimated using StereoInvestigator software (MicroBrightfield, Inc, VT, USA). Scale bar = 20 um. Both Mab1-Fab and Mab2 significantly reduced the formation of damaged axons in the optic nerve, while antibody Mab1 showed a similar trend. [Figure 11A] Protection of photoreceptor neuron loss and retinal function in a mouse light damage model with Mab1 antibody. A shows the light damage model in mice for 7 days following intravitreal (IVT) administration of Mab1 antibody and evaluation of retinal function and histology on day 14. Mice were administered 1 μL of 7.5 mg / mL Mab1 or isotype control antibody via IVT administration on day 7. [Figure 11B](B) Protection of photoreceptor neuron loss and retinal function in a mouse light damage model with Mab1 antibody. (C) Mab1 treatment resulted in a significant reduction in Tunel+ve photoreceptors in the outer nuclear layer of the retina when compared to the isotype control. [Figure 11C] (C) Protection of photoreceptor neuron loss and retinal function in a mouse light damage model with Mab1 antibody. (D) Mab1 treatment resulted in an increase in the number of photoreceptor rows in the outer nuclear layer compared to the isotype control. [Figure 11D] (D) Protection of photoreceptor neuron loss and retinal function in a mouse light damage model with Mab1 antibody. (E) Mab1 antibody treatment resulted in a significant increase in A-wave and B-wave in the retinal view at day 14 when compared with an isotype control antibody. [Figure 12] Free C1q in aqueous humor after a single IVT injection is shown. [Figure 13A] Immunofluorescence (IF) data are shown. [Figure 13B] Immunofluorescence (IF) data are shown in (A) and (B) showing reduced microgliosis in the outer plexiform layer (OPL) (also known as the outer synaptic layer) of the retina 3 days after treatment. Reduced microgliosis is associated with reduced inflammation. [Figure 13C] Immunofluorescence (IF) data are shown. C shows significant preservation of photoreceptor synapses. [Figure 13D] Immunofluorescence (IF) data are shown in Figure 1D, demonstrating significant preservation of cell bodies at 5 days after treatment. [Figure 14] AB show measurable PK and target engagement in the retina. [Figure 15] A is a bar graph showing quantification of immunofluorescence images. B shows immunofluorescence images demonstrating preservation of photoreceptor synapses (BSN marker) upon treatment with C1q inhibitor. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Overview The present disclosure relates generally to compositions and methods for preventing, reducing the risk of developing, or treating inherited retinal diseases (IRDs) (e.g., retinitis pigmentosa, choroideremia, Stargardt disease, cone-rod dystrophy, and Leber congenital amaurosis) or retinal detachment.

[0010] Disclosed herein are recombinant humanized immunoglobulin G (IgG1) antigen-binding fragments (Fabs) that inhibit the classical complement cascade without affecting the lectin pathway or the alternative complement pathway. Anti-C1q Fabs (e.g., FabA, an anti-C1q Fab comprising a heavy chain Fab fragment of SEQ ID NO: 39, and a light chain Fab fragment of SEQ ID NO: 40) are developed for intravitreal (IVT) administration for the treatment of inherited retinal diseases (IRDs) (e.g., retinitis pigmentosa, choroideremia, Stargardt disease, cone-rod dystrophy, and Leber's congenital amaurosis) and retinal detachment. The hypervariable region derived from mouse antibody M1 (Mab1 antibody comprising a heavy chain variable domain of SEQ ID NO: 3 and a light chain variable domain of SEQ ID NO: 7) is described as a human IgG1 Fab fragment construct (FabA). A full-length human IgG4 antibody (Mab2, i.e., an antibody comprising the heavy chain variable domain of SEQ ID NO: 8 and the light chain variable domain of SEQ ID NO: 4) containing the hypervariable region derived from Mab1 was also described. Mab1 and Mab2 as well as their Fabs (Mab1-Fab and Mab2-Fab) were used as surrogate molecules for FabA in pharmacological studies. FabA is a monovalent Fab construct lacking the Fc heavy chain constant domains 2 and 3 (CH2 and CH3) and therefore cannot bind C1q via interaction with the Fc domain. Furthermore, since FabA only has a single antigen-binding arm, it does not show agonistic activity for C1q over a wide range of FabA concentrations.

[0011] The complement cascade is a key component of innate immunity and can be activated via three different pathways: the classical, lectin and alternative pathways. All three pathways lead to the activation of complement component C3, which ultimately leads to immune cell recruitment, inflammation, membrane lysis via the membrane attack complex, and cell death.

[0012] C1q, the initiator of the classical complement cascade, is involved in the initiation and progression of neurodegenerative diseases. Inhibition of C1q may slow neuronal and synaptic damage by blocking the initiation of the classical complement cascade, directly reducing damage to neuronal membranes, and by reducing inflammation resulting from complement activation.

[0013] Mab2-Fab and / or FabA show high affinity binding to human C1q as measured by Biacore (<10 pM) and enzyme-linked immunosorbent assay (ELISA) (40-50 pM, Figure 7). Mab1 binds to the isolated globular head domain of C1q but not to the collagen tail of C1q (determined by ELISA). Consistent with this finding, Mab1 inhibits substrate interactions mediated by the globular head domain of C1q (IgM, C-reactive protein [CRP], and phosphatidylserine) and FabA inhibits the functional interaction of C1q with immunoglobulin M (IgM)-coated red blood cells (RBCs) (blocking hemolysis, Figure 9). Antibody Mab1 specifically recognizes C1q and shows no binding to other complement components (C3b and C5) or to other C1q / tumor necrosis factor (TNF) superfamily members, including TNF, and adiponectin, the protein that shares the highest sequence identity with C1q in the globular head domain. Consistent with these results, FabA does not inhibit the lectin complement pathway initiated by mannose-binding lectin (MBL, another member of the C1q / TNF superfamily) nor the alternative complement pathway (initiated by C3b) (Figure 8).

[0014] 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 term "comprising," the term "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.

[0015] As used herein, administration "in conjunction with" another compound or composition includes simultaneous administration and / or administration at different times. Concurrent administration also encompasses administration as a co-formulation or as separate compositions, including using different dosing frequencies or intervals, and the same or different routes of administration.

[0016] The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody." The term "antibody" is used herein in the broadest sense and specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, antibody fragments, so long as they exhibit biological activity, and antibody derivatives.

[0017] 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 two peptides are paired 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.

[0018] L 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 domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with 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 different classes of immunoglobulins are well known and generally described in, e.g., Abbas et al., Cellular and Molecular Immunology, 4 th This is described in WB Saunders Co., ed., 2000.

[0019] A "full-length antibody" is usually a heterotetrameric glycoprotein of about 150,000 daltons comprising two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at one end a variable domain (V H ) followed by several constant domains. Each light chain has a variable domain (V L ) at its other end and a constant domain, with the light chain constant domain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0020] An "isolated" molecule or cell is a molecule or cell that has been identified and separated from at least one contaminating molecule or cell that is normally associated with the environment in which it was produced. Preferably, an isolated molecule or cell is not associated with all components associated with the environment in which it was produced. An isolated molecule or cell is in a form other than the form or context in which it is found in nature. Thus, an isolated molecule is distinct from a molecule that naturally occurs in a cell; an isolated cell is distinct from a cell that naturally occurs in a tissue, organ, or individual. In some embodiments, the isolated molecule is an anti-C1q antibody of the present disclosure. In other embodiments, the isolated cell is a host cell or hybridoma cell that produces an anti-C1q antibody of the present disclosure.

[0021] An "isolated" antibody is one that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). Preferably, an isolated polypeptide is free of association with all other contaminating components from its production environment. Contaminating components from its production environment, such as those resulting from recombinant transfected cells, are substances that would typically interfere with research, diagnostic or therapeutic uses of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain preferred embodiments, the polypeptide will be purified (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, of the antibody, e.g., as determined by the Lowry method; (2) to a sufficient extent to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue, or preferably silver stain. An isolated antibody includes an antibody in situ in recombinant T cells, since at least one component of the antibody's natural environment is absent. Ordinarily, however, an isolated polypeptide or antibody will be prepared by a process that includes at least one purification step.

[0022] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of an antibody. The heavy and light chain variable domains 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 sites.

[0023] The term "variable" refers to the fact that certain segments of the variable domains vary widely in sequence from one antibody to another. The V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed throughout the variable domains. Rather, it is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Natural 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 within each chain are held in close proximity 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 antibodies (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0024] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous 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., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987) (also referred to herein as Chothia 1987), and MacCallum et al., J. Mol. Biol. 262:732-745 (1996) (the definitions include overlapping or subsets of amino acid residues when compared against each other). Nevertheless, application of either definition to refer to a CDR of an antibody or grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein.

[0025] 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.

[0026] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogenous 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) that may be present in minor amounts. Monoclonal antibodies are highly specific and 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. In addition to their specificity, monoclonal antibodies are advantageous because they are typically synthesized by hybridoma culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as obtained as a substantially homogenous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies used in accordance with the present disclosure can be produced using, for example, hybridoma methods (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14(3):253-260 (1995); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2d ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display techniques (e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., Nature, 352:624-628 (1991)), and the like. al., J.Mol.Biol.222:581-597(1992), Sidhu et al.,J.Mol.Biol.338(2):299-310(2004), Lee et al.,J.Mol.Biol.340(5):1073-1093(2004), Fellouse,Proc.Nat'l Acad.Sci.USA 101(34):12467-472 (2004), and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004)), and techniques for producing human or human-like antibodies in animals that have some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (e.g., WO 1998 / 24893, WO 1996 / 34096, WO 1996 / 33735, WO 1991 / 10741, Jakobovits et al., Proc. Nat'l Acad. Sci. USA 90:2551 (1993), Jakobovits et al., Nature 362:255-258 (1993), Bruggemann et al. Year in Immunol. 7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al., Bio / Technology 10:779-783 (1992), Lonberg et al., Nature 368:856-859 (1994), Morrison, Nature 368:812-813 (1994), Fishwild et al. Nature Biotechnol. 14:845-851 (1996), Neuberger, Nature Biotechnol. 14:826 (1996), and Lonberg and See Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0027] The terms "full length antibody," "intact antibody," and "complete antibody" are used interchangeably to refer to an antibody in substantially intact form, as opposed to an antibody fragment or antibody derivative. In particular, complete antibodies include those having heavy and light chains, including the Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, an intact antibody may have one or more effector functions.

[0028] An "antibody fragment" or "antigen-binding fragment" or "functional fragment" of an antibody comprises a portion of an intact antibody, preferably the F region of an antibody that retains or has the antigen-binding and / or variable region of the intact antibody or modified FcR binding ability. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; and linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062 (1995)). Additional examples of antibody fragments include antibody derivatives, such as single-chain antibody molecules formed from antibody fragments, monovalent antibodies and multispecific antibodies.

[0029] An "antibody derivative" is any construct that contains the antigen-binding region of an antibody. Examples of antibody derivatives include single-chain antibody molecules formed from antibody fragments, monovalent antibodies, and multispecific antibodies.

[0030] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and one residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment contains the entire L chain plus the variable region domain of the H 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 are still capable of cross-linking antigen. The Fab' fragment is H F(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 herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0031] 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 (FcR) found on certain cell types.

[0032] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain and includes native sequence Fc regions and variant Fc regions. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is usually defined as extending from the amino acid residue at position Cys226, or from Pro230, to its 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, an intact antibody composition may include an antibody population with all K447 residues removed, an antibody population without the K447 residue removed, and an antibody population with 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.

[0033] 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.

[0034] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by 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., from about 1 to about 10 amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide, and preferably from about 1 to about 5 amino acid substitutions. 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 therewith, and more preferably at least about 95% homology therewith.

[0035] "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Moreover, a preferred FcR binds an IgG antibody (gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors, where FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif ("ITIM") in its cytoplasmic domain. (See, e.g., M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. FcRs may increase the serum half-life of an antibody.

[0036] The in vivo binding to FcRn and serum half-life of human FcRn high affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates to which the polypeptides having variant Fc regions are administered. WO2004 / 42072 (Presta) describes antibody variants with improved or reduced binding to FcR. See also, for example, Shields et al., J.Biol.Chem.9(2):6591-6604(2001).

[0037] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. The folding of these two domains results in six hypervariable loops (three loops each from the H chain and L chain) that provide amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the entire binding site.

[0038] A "single-chain Fv", also abbreviated to "sFv" or "scFv", is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected in a single polypeptide chain. Preferably, the sFv polypeptide comprises a V H Domain and V L It further comprises a polypeptide linker between the domains which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0039] The term "diabody" refers to a diabody that is a peptide that binds V domains together to achieve inter-chain but not intra-chain V domain pairing, thereby resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. H Domain and V L This refers to small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with a short linker (approximately 5-10 residues) between the domains. Bispecific diabodies are the VFv fragments of two antibodies. H and V LDiabodies are heterodimers of two "crossover" sFv fragments in which the domains are present on different polypeptide chains. Diabodies are described in more detail, for example, in EP 404,097, WO 1993 / 011161, WO / 2009 / 121948, WO / 2014 / 191493, Hollinger et al., Proc. Nat'l Acad. Sci. USA 90:6444-48 (1993).

[0040] As used herein, "chimeric antibody" refers to antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical 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 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."

[0041] "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 the recipient antibody or in the donor antibody. These modifications may be made to further improve 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, but 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 be no more than six in the H chain and no more than three in the L chain. The humanized antibody will also optionally 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.

[0042] A "human antibody" is an antibody having an amino acid sequence that corresponds 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 that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. The methods described in 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); 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 xenomice, that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled (see, e.g., U.S. Pat. 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.

[0043] As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops. Generally, an antibody contains six HVRs, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). In natural antibodies, H3 and L3 exhibit the highest diversity among these six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camelid antibodies consisting only of heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct Biol. 3:733-736 (1996).

[0044] Several HVR delineations are used and encompassed herein. The 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)). The AbM HVRs represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" HVRs are based on an analysis of available complex crystal structures. Residues from each of these HVRs are listed below. [Table 1]

[0045] 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.

[0046] "Framework" or "FR" residues are those variable domain residues other than HVR residues as herein defined.

[0047] The phrases "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system used for the heavy or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or HVR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined for a given antibody by matching the sequence of the antibody with the "standard" Kabat numbered sequence at the regions of homology.

[0048] 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).

[0049] An "acceptor human framework", as used herein, is a framework that comprises the amino acid sequence of a VL framework or a 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 that, 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 only at 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 the human consensus framework sequence.

[0050] 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.

[0051] 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 1-2 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.

[0052] An "affinity matured" antibody is one that has one or more changes in one or more HVRs thereof that result in an improvement in the affinity of the antibody for antigen compared to a parent antibody that does not have those change(s). In some embodiments, an affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies are generated by procedures known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and / or framework residues has been described, for example, in Barbas et al. Proc Nat. Acad. Sci. USA 91:3809-3813 (1994), Schier et al. Gene 169:147-155 (1995), Yelton et al. J. Immunol. 155:1994-2004 (1995), Jackson et al., J. Immunol. 154(7):3310-9 (1995), and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0053] As used herein, the term "specifically recognize" or "specifically bind" refers 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 an antibody 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 an association constant of 100 or more. 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, for example, 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.

[0054] "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 may be substituted for isoleucine or valine. Other amino acids that may often be substituted for one another include, but are not limited to: - 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 -Cysteine ​​and methionine (amino acids with sulfur-containing side chains)

[0055] Degrees 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).

[0056] As used herein, an "interaction" between a complement protein 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 an 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 an interaction" between two proteins if the antibody or a fragment thereof binds to one of the two proteins.

[0057] A "blocking," "antagonist," "inhibitory," or "neutralizing" antibody is an antibody that inhibits or reduces one or more biological activities of the antigen to which it binds, e.g., interaction with one or more proteins. In some embodiments, a blocking, antagonist, inhibitory, or "neutralizing" antibody substantially or completely inhibits one or more biological activities or interactions of an antigen.

[0058] The term "inhibitor" refers to a compound that has the ability to inhibit the biological function of a target biomolecule, e.g., an mRNA or a protein, whether by decreasing the activity or expression of the target biomolecule. An inhibitor can be an antibody, a small molecule, or a nucleic acid molecule. The term "antagonist" refers to a compound that binds to a receptor and blocks or attenuates the biological response of the receptor. The term "inhibitor" can also refer to an "antagonist."

[0059] Antibody "effector functions" refer to the biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype.

[0060] As used herein, the term "affinity" refers to the equilibrium constant for the reversible binding of two substances (e.g., an antibody and an antigen), expressed as the dissociation constant (KD). The affinity can be at least 1-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 20-fold higher, at least 30-fold higher, at least 40-fold higher, at least 50-fold higher, at least 60-fold higher, at least 70-fold higher, at least 80-fold higher, at least 90-fold higher, at least 100-fold higher, or at least 1,000-fold higher, or more, than the affinity of the antibody for an unrelated amino acid sequence. The affinity of the antibody for the target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM), or more. As used herein, the term "avidity" refers to the resistance of a complex of two or more substances to dissociation upon dilution. The terms "immunoreactive" and "preferentially bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0061] The term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. For example, a subject anti-C1q antibody specifically binds to an epitope within the complement C1q protein. "Specific binding" refers to binding that is at least about 10 -7 M or more, e.g., 5×10 -7 M, 10 -8 M, 5×10 -8 "Non-specific binding" refers to binding with an affinity of about 10 -7 Affinity of less than M, e.g., 10 -6 M, 10 -5 M, 10 -4 It refers to binding with an affinity of M or similar.

[0062] "k onThe term "rate constant" as used herein is intended to refer to the rate constant for the association of an antibody to an antigen.

[0063] "k off The term, as used herein, is intended to refer to the rate constant for dissociation of an antibody from the antibody / antigen complex.

[0064] "K D The term "as used herein" is intended to refer to the equilibrium dissociation constant of an antibody-antigen interaction.

[0065] 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 the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps as 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 necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0066] A "biological sample" encompasses a variety of sample types obtained from an individual and may be used in diagnostic or monitoring assays. The definition encompasses blood and other liquid samples of biological origin, solid tissue samples, such as biopsy specimens or tissue cultures or cells derived therefrom, and their progeny. The definition also includes samples that have been manipulated in any way after their procurement, for example, by treatment with reagents, solubilization, or enrichment for certain components, such as polynucleotides. The term "biological sample" encompasses clinical samples, and also includes cultured cells, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood fractions, such as plasma and serum, and the like. The term "biological sample" also encompasses solid tissue samples, tissue culture samples, and cell samples.

[0067] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule that is normally associated with the environment in which it is produced. Preferably, an isolated nucleic acid is free of association with all components associated with the environment in which it is produced. The isolated nucleic acid molecules encoding the polypeptides and antibodies herein are in a form other than the form or setting in which they are found in nature. Thus, isolated nucleic acid molecules are distinct from any nucleic acid encoding the polypeptides and antibodies herein that naturally occur in a cell.

[0068] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Additionally, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors," or simply "expression vectors." Typically, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably, as the plasmid is the most commonly used form of vector.

[0069] "Polynucleotide", or "nucleic acid", as used interchangeably herein, refers to a polymer of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. A polynucleotide can include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of nucleotides can be separated by non-nucleotide components. A polynucleotide can include modification(s) that are generated post-synthetically, such as conjugation to a label. Other types of modifications include, for example, "caps" which replace one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant sites such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalating agents (e.g., acridine, psoralen, etc.), those containing chelating agents (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Additionally, any of the hydroxyl groups normally present in the sugar may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to generate additional bonds to additional nucleotides, or conjugated to solid or semi-solid supports. The 5' and 3' terminal OH may be phosphorylated or replaced with amines or organic capping group moieties of 1-20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.Polynucleotides may contain analog forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acrylic acid analogs, and basic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl, optionally containing an ether (-O-) linkage. Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0070] "Host cell" includes an individual cell or cell culture that can be or has been a recipient for a vector(s) for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, sudden, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of the present disclosure.

[0071] As used herein, "carrier" includes pharma- ceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations used. Often, physiologically acceptable carriers are aqueous pH buffers. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0072] The term "prevent" is art-recognized and when used in reference to conditions such as inherited retinal diseases (IRDs) (e.g., retinitis pigmentosa / rod-cone dystrophy, choroideremia, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis, X-linked RP, and Usher syndrome) or retinal detachment or related conditions, compared to patients not receiving the therapy.

[0073] 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, such as chimpanzees, and other ape and monkey species; farm animals, such as cows, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs, and the like. The term does not denote a particular age or sex.

[0074] As used herein, the term "treating" or "treatment" includes reducing, arresting, or reversing the symptoms, clinical signs, or underlying pathology of a condition, stabilizing or ameliorating the subject's condition, or reducing the likelihood that the subject's condition will worsen to the same extent as if the subject had not received treatment.

[0075] "Recovery" refers to the act of returning to a normal or healthy state. Recovery can be partial (e.g., when a subject returns to a state below a normal or healthy state) or complete (e.g., when a subject returns to a state that is the same or nearly the same as a normal or healthy state). An example of a normal or healthy state is the patient's vision before retinal detachment.

[0076] "Improving vision" refers to the act of improving visual ability or condition, including improving visual acuity, sensitivity, and / or range of visual field compared to before treatment.

[0077] The term "therapeutically effective amount" of a compound in the context of a method of treating a subject 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), relieves symptoms, ameliorates pathology, or delays the onset of a disease state, for example, in accordance with clinically accepted standards for treating a disorder or condition, or for cosmetic purposes, at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutically effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the patient, as well as the ability of the antibody to elicit a desired response in the individual.

[0078] As used herein, an individual "at risk" of developing a particular disease, disorder, or condition may or may not exhibit detectable disease or disease symptoms, and may or may not exhibit detectable disease or disease symptoms prior to the treatment methods described herein. "At risk," as known in the art, indicates that an individual has one or more risk factors, which are measurable parameters that correlate with the development of a particular disease, disorder, or condition. Individuals who have one or more of these risk factors are more likely to develop a particular disease, disorder, or condition than individuals who do not have one or more of these risk factors.

[0079] "Chronic" administration refers to the administration of a pharmaceutical agent(s) continuously, as opposed to an acute mode, so as to maintain an initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration refers to treatment that is not administered continuously without interruption, but rather is cyclic / periodic in nature.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference, including, but not limited to, methods and / or materials related to which the publications are cited, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (FMA Usubel, et al. eds., (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)), Oligonucleotide Synthesis (MJ Gait, ed., 1984), Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Animal Cell Culture (RIFreshney), ed., 1987), Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGriffiths, and D.G. Newell, eds., 1993-8), J. Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987), PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994), Current Protocols in Immunology (JEColigan et al., eds., 1991), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: A Practical Approach (D. Catty., ed., IRL Press, 1988-1989), Monoclonal Antibodies: A Practical The present invention discloses and explains widely used methodologies described in: Antibodies: A Laboratory Manual (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JBLippincott Company, 1993).

[0081] 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.

[0082] Full-length antibodies can be prepared by the use of recombinant DNA engineering techniques. Such engineered versions include, for example, those created from natural antibody variable regions by insertions, deletions or changes within or to the amino acid sequence of the natural antibody. Particular examples of this type include those engineered variable region domains that contain at least one CDR from a first 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 a desired portion of DNA encoding a full-length antibody. DNA encoding a chimerized antibody can be prepared by recombining DNA encoding substantially or exclusively human constant regions and DNA encoding variable regions substantially or exclusively derived from variable region sequences of a mammal other than 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 and DNA encoding CDRs substantially or exclusively derived from a mammal other than human.

[0083] Suitable sources of DNA molecules encoding an antibody include cells, such as hybridomas, that express the full-length antibody. For example, an antibody can be isolated from a host cell that expresses an expression vector encoding the heavy and / or light chain of the antibody.

[0084] Antibody fragments, including but not limited to Fab fragments, and / or antibody derivatives can also be prepared by the use of recombinant DNA modification techniques, including the manipulation and re-expression of DNA encoding the 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, the C H 1 domain translation is terminated at the interchain cysteine. H PCR is used to generate antibody fragments by introducing a stop codon immediately following the codon encoding the interchain cysteine ​​of antibody C1. Methods for designing suitable PCR primers are well known in the art and can be found in H The sequences of the 1 domains are readily available. In some embodiments, a stop codon can be introduced using site-directed mutagenesis techniques.

[0085] The 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.

[0086] The antibodies of the present disclosure may 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 characterized by: (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 red blood cell lysis; (6) reduced erythrophagocytosis; (7) reduced dendritic cell infiltration; (8) inhibition of complement mediated red blood cell 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 erythrocyte infiltration; (18) reduced erythrocyte lysis; (19) reduced lymphocyte infiltration; (20) reduced macrophage infiltration; (21) reduced antibody deposition; (22) reduced erythrocyte lysis; (23) reduced erythrocyte lysis; (24) reduced erythrocyte lysis; (25) reduced erythrocyte lysis; (26) reduced erythrocyte lysis; (27) reduced erythrocyte lysis; (28) reduced erythrocyte lysis; (29) reduced erythrocyte infiltration; (30) reduced erythrocyte lysis; (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 Decreased neutrophil infiltration, (13) decreased platelet phagocytosis, (14) decreased platelet lysis, (15) improved graft survival, (16) decreased macrophage-mediated phagocytosis, (17) decreased autoantibody-mediated complement activation, (18) decreased red blood cell destruction in transfusion reactions, (19) decreased alloantibody-induced red blood cell lysis, (20) decreased hemolysis in transfusion reactions, (21) decreased alloantibody-mediated platelet lysis, (22) improved anemia, (23) decreased eosinophilia, (24 ) reduced C3 deposition on red blood cells (e.g., reduced C3b, iC3b, etc. deposition on RBCs), (25) reduced C3 deposition on platelets (e.g., reduced C3b, iC3b, etc. deposition on platelets), (26) reduced anaphylatoxin production, (27) reduced autoantibody-mediated rash formation, (28) reduced autoantibody-induced lupus erythematosus, (29) reduced red blood cell destruction in transfusion reactions, (30) reduced platelet lysis in transfusion reactions, (31) obesity These include decreased cellular 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.

[0087] 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 the CH50 hemolysis. In some embodiments, the antibody is capable of neutralizing 50%, 60%, 70%, 80, 90%, or 100% of the CH50 hemolysis. The antibody may also be capable of neutralizing at least 50% of the 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.

[0088] Other in vitro assays for measuring complement activity include ELISA assays for the measurement of cleavage products of complement components or complexes formed during complement activation. Complement activation via the classical pathway can be measured by following the levels of C4d and C4 in serum. 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.

[0089] The antibodies of the present disclosure may be monoclonal, polyclonal, recombinant, humanized, human, chimeric, multispecific, antibody fragments thereof, or derivatives thereof. In some embodiments, the antibody is a humanized antibody.

[0090] The 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.

[0091] In some embodiments, the antibody is a human monoclonal antibody that may be prepared, expressed, generated or isolated by recombinant means, such as (a) an antibody isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal of 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, generated or isolated by any other means, including splicing of human immunoglobulin gene sequences to 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 V regions of the recombinant antibody. H and V L The amino acid sequence of the region is 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.

[0092] 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 can be used for immunization, such as non-human primates, transgenic mice expressing human immunoglobulins, and severe combined immunodeficient (SCID) mice engrafted with human B lymphocytes.

[0093] Polyclonal and monoclonal antibodies are naturally produced as immunoglobulin (Ig) molecules in the immune system's response to pathogens. The predominant form in human serum, the IgG1 molecule of about 150 kDa, has a concentration of 8 mg / ml and is composed of two identical heavy chains of about 50 kDa and two identical light chains of about 25 kDa.

[0094] Hybridomas can be produced by conventional procedures by fusing B lymphocytes from immunized animals with myeloma cells. Anti-C1q antibodies can also be produced by screening recombinant single-chain Fv or Fab libraries from human B lymphocytes in a phage display system. The specificity of MAbs for human C1q can be tested by enzyme-linked immunosorbent assay (ELISA), Western immunoblotting, or other immunochemical techniques.

[0095] 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 show specific inhibitory activity against the classical complement pathway are cloned by limiting dilution. The antibodies are purified for characterization of specificity against human C1q by the assay described above.

[0096] Anti-complement C1q antibodies The anti-C1q antibodies disclosed herein are potent inhibitors of C1q and can be dosed 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. The results obtained with the anti-C1q antibodies disclosed herein in animal studies can be readily advanced to the clinic using humanized or human antibodies, as well as fragments and / or derivatives thereof.

[0097] C1q is a large multimeric protein of 460 kDa consisting of 18 polypeptide chains (6 C1q A chains, 6 C1q B chains, and 6 C1q C chains). The C1r and C1s complement proteins bind to the C1q tail region to form the C1 complex (C1qr2s2).

[0098] 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 human, mouse, rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pig.

[0099] 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 )

[0100] The anti-C1q antibodies disclosed herein can inhibit C1 complex formation.

[0101] As used herein, "complement factor C1q" refers to both the wild-type sequence and the naturally occurring variant sequence.

[0102] 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 PPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA. C1q, chain B (Homo sapiens), accession number protein Database: NP_000482.3; GenBank number: NM_000491.3: >gi|87298828|ref|NP_000482.3|Complement C1q Subcomponent Subunit B Precursor [Homo sapiens] (SEQ ID NO:2) 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:3) MDVGPSSLPHLGLKLLLLLLLLPLRGQANTGCYGIPGMPGLPGAPGKDGYDGLPGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGIPGEPGEEGRYKQKFQSVFT VTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD.

[0103] Thus, an anti-C1q antibody of the disclosure may bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of a C1q protein. In some embodiments, an anti-C1q antibody of the disclosure binds to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of human C1q or a homolog thereof, 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.

[0104] All sequences listed in the next 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.

[0105] Light and heavy chain variable domain sequences of antibody M1 (Mab1) The nucleic acid and amino acid sequences encoding the light 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:

[0106] TIFF2025515485000002.tif20170

[0107] 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).

[0108] The amino acid sequence of the heavy chain variable domain of antibody M1 is:

[0109] TIFF2025515485000003.tif29170

[0110] 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).

[0111] The nucleic acid sequence encoding the light chain variable domain was determined to be: GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAACAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTGCAA TCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAACATAATGAATACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 12).

[0112] The nucleic acid sequence encoding the heavy chain variable domain was determined to be: CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGTCTTCTGGCTACCATTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGTGATTCATCCTAATAGTGGTAGTATTAACTACAATGAG AAGTTCGAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCGGCGGTCTATTATTGTGCAGGAGAGAGAGATTCTACGGAGGTTCTCCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 13).

[0113] Mab1-Fab is the Fab of the Mab1 (M1) antibody.

[0114] Mab3 is a murine anti-C1q antibody derived from the Mab1 antibody and optimized for mouse experiments, and Mab3-Fab is the Fab of the Mab3 antibody.

[0115] 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 2]

[0116] A hybridoma cell line (mouse hybridoma C1qM1 7788-1(M)051613) which produces the M1 antibody has been deposited with the ATCC under conditions which ensure that access to the culture will be available during the pendency of the patent application and for 30 years, or 5 years after the latest claim, or until 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 of this application, or progeny thereof, are filed. It should be understood, however, that the availability of the deposit does not constitute a license to practice the invention in derogation from patent rights granted by governmental action.

[0117] Disclosed herein is a method of administering an anti-C1q antibody comprising a light chain variable domain and a heavy chain variable domain. The antibody may bind at least human C1q, mouse C1q, or rat C1q. The antibody may be a humanized antibody, a chimeric antibody, or a human antibody. The antibody may be a monoclonal antibody, an antibody fragment thereof, and / or an antibody derivative thereof. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment, such as a Fab fragment. The light chain variable domain 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 heavy chain variable domain 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.

[0118] 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.

[0119] 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).

[0120] Disclosed herein is a method for administering an anti-C1q antibody that inhibits the interaction between C1q and autoantibodies. In a preferred embodiment, the anti-C1q antibody causes the clearance of C1q from the circulation or tissues.

[0121] In some embodiments, the anti-C1q antibodies of the present disclosure inhibit the interaction between C1q and C1s. In some embodiments, the anti-C1q antibodies inhibit the interaction between C1q and C1r. In some embodiments, the anti-C1q antibodies inhibit the interaction between C1q and C1s and between C1q and C1r. In some embodiments, the anti-C1q antibodies inhibit the interaction between C1q and another antibody, e.g., an autoantibody. In preferred embodiments, the anti-C1q antibodies cause the clearance of C1q from the circulation or tissues. In some embodiments, the anti-C1q antibodies inhibit the respective interactions with a stoichiometry of less than 2.5:1, 2.0:1, 1.5:1, or 1.0:1. In some embodiments, the C1q antibodies inhibit interactions such as the C1q-C1s interaction at approximately equimolar concentrations of C1q and anti-C1q antibodies. In other embodiments, the anti-C1q antibody is less than 20:1, less than 19.5:1, less than 19:1, less than 18.5:1, less than 18:1, less than 17.5:1, less than 17:1, less than 16.5:1, less than 16:1, less than 15.5:1, less than 15:1, less than 14.5:1, less than 14:1, less than 13.5:1, less than 13:1, less than 12.5:1, less than 12:1, less than 11.5:1, less than 11:1 , 10.5:1, 10:1, 9.5:1, 9:1, 8.5:1, 8:1, 7.5:1, 7:1, 6.5:1, 6:1, 5.5:1, 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2.0:1, 1.5:1, or 1.0:1. In certain embodiments, the anti-C1q antibody binds to C1q with a binding stoichiometry ranging from 20:1 to 1.0:1 or less than 1.0:1. In certain embodiments, the anti-C1q antibody binds to C1q with a binding stoichiometry ranging from 6:1 to 1.0:1 or less than 1.0:1. In certain embodiments, anti-C1q antibodies bind to C1q with a binding stoichiometry ranging from 2.5:1 to 1.0:1 or less than 1.0:1. In some embodiments, anti-C1q antibodies inhibit the interaction between C1q and C1r, or between C1q and C1s, or between C1q and both C1r and C1s.In some embodiments, the anti-C1q antibody inhibits the interaction between C1q and C1r, between C1q and C1s, and / or between C1q and both C1r and C1s. In some embodiments, the anti-C1q antibody binds to the A chain of C1q. In other embodiments, the anti-C1q antibody binds to the B chain of C1q. In other embodiments, the anti-C1q antibody binds to the C chain of C1q. In some embodiments, the anti-C1q antibody binds to the A chain of C1q, the B chain of C1q, and / or the C chain of C1q. In some embodiments, the anti-C1q antibody binds to the globular domain of the A chain, the B chain, and / or the C chain of C1q. In other embodiments, the anti-C1q antibody binds to the collagen-like domain of the A chain, the B chain, and / or the C chain of C1q.

[0122] When an antibody of the present disclosure inhibits an interaction between two or more complement factors, for example, an interaction between C1q and C1s or an interaction between C1q and C1r, the interaction that occurs in the presence of the antibody may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to a control in which an antibody of the present disclosure is not present. In some embodiments, an antibody of the present disclosure reduces an interaction between two or more complement factors by 50%, 60%, 70%, 80%, 90%, or 100%. In certain embodiments, the interaction that occurs in the presence of the antibody is reduced by an amount ranging from at least 30% to at least 99% compared to a control in which an antibody of the present disclosure is not present.

[0123] In some embodiments, an antibody of the present disclosure inhibits C2 or C4 cleavage by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or an amount ranging from at least 30% to at least 99%, compared to a control in which no antibody of the present disclosure is present. Methods for measuring C2 or C4 cleavage are well known in the art. The EC of an antibody of the present disclosure with respect to C2 or C4 cleavage can be determined by the following methods: 50Values ​​may be less than 3 μg / ml, 2.5 μg / ml, 2.0 μg / ml, 1.5 μg / ml, 1.0 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.1 μg / ml, 0.05 μg / ml, In some embodiments, the antibodies of the disclosure inhibit C2 or C4 cleavage at approximately equimolar concentrations of C1q and the respective anti-C1q antibodies.

[0124] In some embodiments, an antibody of the disclosure inhibits autoantibody- and complement-dependent cytotoxicity (CDC) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or an amount ranging from at least 30% to at least 99%, relative to a control lacking the antibody of the disclosure. 50 Values ​​can be less than 3 μg / ml, 2.5 μg / ml, 2.0 μg / ml, 1.5 μg / ml, 1.0 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.1 μg / ml, 0.05 μg / ml.

[0125] In some embodiments, the antibodies of the present disclosure inhibit complement-dependent cell-mediated cytotoxicity (CDCC) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, or an amount ranging from at least 30% to at least 99%, relative to a control lacking the antibody of the present disclosure. Methods for measuring CDCC are well known in the art. With respect to CDCC inhibition, the EC 50 Values ​​may be less than 3 μg / ml, 2.5 μg / ml, 2.0 μg / ml, 1.5 μg / ml, 1.0 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.1 μg / ml, 0.05 μg / ml, In some embodiments, the antibodies of the disclosure inhibit CDCC but do not inhibit antibody-dependent cellular cytotoxicity (ADCC).

[0126] Humanized anti-complement C1q antibody 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.

[0127] All sequences referred to in the following 16 paragraphs are incorporated by reference from U.S. Patent Application Serial No. 14 / 933,517, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0128] In some embodiments, the human heavy chain constant region is a human IgG4 heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% homology to SEQ ID NO: 47. The human IgG4 heavy chain constant region may comprise an Fc region having one or more modifications and / or amino acid substitutions according to Kabat numbering. In such cases, the Fc region comprises an amino acid substitution of leucine to glutamate at position 248, such a substitution inhibits the Fc region from interacting with an Fc receptor. In some embodiments, the Fc region comprises an amino acid substitution of serine to proline at position 241, such a substitution prevents arm switching in the antibody.

[0129] The amino acid sequence of the human IgG4 (S241P L248E) heavy chain constant domain is: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 47).

[0130] The antibody may comprise a heavy chain variable domain and a light chain variable domain, wherein the heavy chain variable domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 31-34, or an amino acid sequence having at least about 90% homology to an amino acid sequence selected from any one of SEQ ID NOs: 31-34. In certain such embodiments, the light chain variable domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 35-38, or an amino acid sequence having at least about 90% homology to an amino acid sequence selected from any one of SEQ ID NOs: 35-38.

[0131] The amino acid sequence of the heavy chain variable domain variant 1 (VH1) is:

[0132] TIFF2025515485000005.tif31170The hypervariable regions (HVRs) of VH1 are shown in bold and underlined text.

[0133] The amino acid sequence of the heavy chain variable domain variant 2 (VH2) is:

[0134] TIFF2025515485000006.tif28170 Hypervariable regions (HVRs) of VH2 are shown in bold and underlined text.

[0135] The amino acid sequence of the heavy chain variable domain variant 3 (VH3) is:

[0136] TIFF2025515485000007.tif29170The hypervariable regions (HVRs) of VH3 are shown in bold and underlined text.

[0137] The amino acid sequence of the heavy chain variable domain variant 4 (VH4) is:

[0138] TIFF2025515485000008.tif30170The hypervariable regions (HVRs) of VH4 are shown in bold and underlined text.

[0139] The amino acid sequence of the kappa light chain variable domain variant 1 (Vκ1) is:

[0140] TIFF2025515485000009.tif25170The hypervariable regions (HVRs) of Vκ1 are shown in bold and underlined text.

[0141] The amino acid sequence of the kappa light chain variable domain variant 2 (Vκ2) is:

[0142] TIFF2025515485000010.tif26170The hypervariable regions (HVRs) of Vκ2 are shown in bold and underlined text.

[0143] The amino acid sequence of the kappa light chain variable domain variant 3 (Vκ3) is:

[0144] TIFF2025515485000011.tif26170The hypervariable regions (HVRs) of Vκ3 are shown in bold and underlined text.

[0145] The amino acid sequence of the kappa light chain variable domain variant 4 (Vκ4) is:

[0146] TIFF2025515485000012.tif22170The hypervariable regions (HVRs) of Vκ4 are shown in bold and underlined text.

[0147] The antibody may comprise a light chain variable domain amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NOs: 35-38, while retaining HVR-L1 RASKSINKYLA (SEQ ID NO: 5), HVR-L2 SGSTLQS (SEQ ID NO: 6), and HVR-L3 QQHNEYPLT (SEQ ID NO: 7). The antibody may comprise a heavy chain variable domain amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NOs: 31-34, while retaining preferably HVR-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO: 10), and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0148] 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.

[0149] The full length antibody Mab2 comprises 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.

[0150] In some embodiments, the humanized anti-C1q antibody of the present disclosure comprises a heavy chain variable region containing a Fab region and a heavy chain constant region containing an Fc region, wherein the Fab region specifically binds to 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 the humanized anti-C1q antibody of the present disclosure comprises an amino acid substitution at position 248 according to the Kabat numbering convention or at a position corresponding to position 248 according to the Kabat numbering convention, and / or at position 241 according to the Kabat numbering convention or at a position corresponding to position 241 according to the Kabat numbering convention. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 inhibits the 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 an arm switch 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 an amino acid sequence of SEQ ID NO:47, or an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homology to the amino acid sequence of SEQ ID NO:47.

[0151] Anti-C1q Fab fragment (e.g., FabA) Before the advent of recombinant DNA technology, proteolytic enzymes (proteases) that cleave the polypeptide sequence were used to break down the structure of antibody molecules and determine which parts of the molecule are responsible for their various functions. Limited digestion with the protease papain cleaves an antibody molecule into three fragments. Two of the fragments, known as Fab fragments, are identical and contain antigen-binding activity. The Fab fragments correspond to two identical arms of the antibody molecule, each of which contains the V of the heavy chain. H and C H The Fab fragment consists of a complete light chain paired with an Fc domain. The other fragment was initially observed to contain no antigen-binding activity but was easily crystallized, and for this reason was named the Fc fragment (fragment crystallizable). When comparing Fab molecules with IgG molecules, Fabs were found to be superior to IgG for certain in vivo applications due to their higher mobility and tissue penetration capabilities, their reduced circulating half-life, their ability to bind antigen monovalently without mediating antibody effector functions, and their lower immunogenicity.

[0152] Fab molecules contain the constant domain C H 2 and C H It is an artificial approximately 50 kDa fragment of an Ig molecule with the heavy chain shortened by 3. Two heterophils (V L -V H and C L -C H 1) Domain interactions underlie the structure of the two chains of the Fab molecule, which is L and C. H 1. Fab and IgG have six complementarity determining regions (CDRs), three each of which is V L and V H The CDRs define the hypervariable antigen-binding site of the antibody. The highest sequence variation is found in LCDR3 and HCDR3, which in the natural immune system are V and VD, respectively. L and J L Gene or V H , DH and J H They are generated by genetic rearrangement. LCDR3 and HCDR3 typically form the core of the antigen-binding site. The conserved regions connecting and presenting the six CDRs are called framework regions. In the three-dimensional structure of the variable domain, the framework regions form a sandwich of two opposing antiparallel β-sheets linked on the outside by the hypervariable CDR loops and on the inside by conserved disulfide bridges. This unique combination of stability and versatility of the antigen-binding sites of Fab and IgG underscores their success in clinical practice for the diagnosis, monitoring, prevention, and treatment of diseases.

[0153] All anti-C1q antibody Fab fragment sequences are incorporated by reference from US patent application Ser. No. 15 / 360,549, which is incorporated by reference herein for the antibodies and related compositions it discloses.

[0154] 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 ), wherein the anti-C1q antibody Fab fragment binds to the C1q protein and comprises six complementarity determining regions (CDRs) (three of which are V L and V H The heavy chain of the antibody Fab fragment is truncated after the first heavy chain domain of IgG1 (SEQ ID NO: 39) and comprises the following amino acid sequence:

[0155] TIFF2025515485000013.tif43170

[0156] The complementarity determining regions (CDRs) of SEQ ID NO:39 are shown in bold and underlined text.

[0157] The light chain domain of the antibody Fab fragment comprises the following amino acid sequence (SEQ ID NO:40):

[0158] TIFF2025515485000014.tif41170

[0159] The complementarity determining regions (CDRs) of SEQ ID NO:40 are shown in bold and underlined text.

[0160] 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.

[0161] Mab1-Fab is the Fab of the Mab1 (M1) antibody.

[0162] Mab2-Fab is the Fab of the Mab2 antibody.

[0163] Mab3-Fab is the Fab of the Mab3 antibody.

[0164] Nucleic Acids, Vectors and Host Cells Antibodies suitable for use in the methods of the present disclosure may be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In some embodiments, an isolated nucleic acid having a nucleotide sequence encoding any of the antibodies of the present disclosure is 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 containing (1) the V of an antibody. L / C L Amino acid sequence containing and V of antibody H / C H (2) a vector containing a nucleic acid encoding an amino acid sequence containing the V L / C L A first vector containing a nucleic acid encoding an amino acid sequence containingH / C H The host cell may contain (e.g., be transduced with) a second vector containing a nucleic acid encoding an amino acid sequence containing 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.

[0165] A method for producing an anti-C1q antibody is disclosed herein. The method includes culturing a host cell of the present disclosure that contains a nucleic acid encoding an anti-C1q antibody under conditions suitable for the expression of the antibody. In some embodiments, the antibody is then recovered from the host cell (or the host cell culture medium).

[0166] For recombinant production of the humanized anti-C1q antibodies of the present disclosure, nucleic acids encoding the antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0167] Suitable vectors containing a nucleic acid sequence encoding an antibody of the present disclosure, or any of its fragment polypeptides (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 large number of cloning vectors available in the art. While the cloning vector selected can vary depending on the host cell intended to be used, useful cloning vectors usually have the ability to replicate autonomously, may possess a single target for a particular restriction endonuclease, and / or may have a gene for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Stratagene, and Invitrogen.

[0168] The vector containing the nucleic acid of interest can be introduced into the host cell by any of a number of suitable means, including electroporation, transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; particle bombardment; lipofection; and infection (e.g., the vector is an infectious agent such as vaccinia virus). The choice of introducing the 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 the anti-C1q antibody of the present disclosure.

[0169] 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 when glycosylation and Fc effector functions are not required. Expression of antibody fragments and polypeptides in bacteria is described in, for example, 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, which describe the expression of antibody fragments in E. coli. In other embodiments, the antibodies of the 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 Serial No. 14 / 269,950, U.S. Patent No. 8,981,071, Eur J Biochem. 1991 Jan 1;195(1):235-42). After expression, the antibodies can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0170] Pharmaceutical Compositions and Administration The anti-C1q antibodies (eg, FabA) of the present disclosure may be administered in the form of a pharmaceutical composition.

[0171] Therapeutic formulations of the antibodies, antibody fragments and / or antibody derivatives of the present disclosure may be prepared in the form of a lyophilized formulation or an aqueous solution for storage by mixing the antibody having the desired purity with any pharma- ceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.

[1980] ). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as Examples of suitable surfactants include serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0172] Lipofections or liposomes can also be used to deliver the antibody or antibody fragment, or antibody derivative, to cells, preferably an epitope or minimal fragment that specifically binds to the binding domain of the target protein.

[0173] The antibodies may also be incorporated into microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0174] The formulations used for administration can be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0175] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid ester, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for over 100 days, certain hydrogels release proteins for shorter periods of time.

[0176] The antibodies, antibody fragments and / or antibody derivatives and compositions of the disclosure are typically administered by intravitreal administration.

[0177] The pharmaceutical composition may also include a pharma- ceutically acceptable non-toxic carrier of a diluent, defined as a vehicle commonly used to formulate a pharmaceutical composition for animal or human administration, depending on the desired formulation. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, physiological saline, PBS, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may include other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, excipients, and the like. The composition may also include additional substances for approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents.

[0178] The composition may also include any of a variety of stabilizing agents, such as, for example, antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide may be complexed with a variety of well-known compounds that improve the in vivo stability of the polypeptide or otherwise improve its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, improve other pharmacokinetic and / or pharmacodynamic properties, or improve solubility or uptake). Examples of such modifying or complexing agents include sulfates, gluconates, citrates, and phosphates. The polypeptides of the composition may also be complexed with molecules that improve their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids. Further guidance regarding suitable formulations for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985). For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990).

[0179] Toxicity and therapeutic efficacy of an active ingredient can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, including, for example, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred.

[0180] Data obtained from cell culture and / or animal studies and / or human clinical trials can be used in formulating a range of dosages for humans.The dosage of active ingredient typically falls within a range of circulating concentrations that includes the ED50 with low toxicity.Dosages can vary within this range depending on the dosage form used and the route of administration utilized.

[0181] The components used to formulate pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, usually at least analytical grade, more typically at least pharmaceutical grade). Furthermore, compositions intended for parenteral use are usually sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is typically substantially free of any potentially toxic substances, particularly any endotoxins, that may be present during the synthesis or purification process. Compositions for parenteral administration are also typically substantially isotonic and produced under GMP conditions.

[0182] The compositions of the present disclosure may be administered using any medically appropriate procedure, for example, intravitreal injection.

[0183] Treatment method The role of complement in photoreceptor retinal diseases has been reported in multiple preclinical models of photoreceptor degeneration, and genetic or pharmacological inhibition of the classical complement pathway resulted in increased photoreceptor survival and improved retinal function. However, the mechanisms by which C1q and the classical complement pathway promote photoreceptor degeneration are not fully understood. Based on the described role of C1q in microglia-mediated synaptic pruning in CNS development and disease, C1q may tag photoreceptor synapses in retinal degenerative disorders and contribute to neuronal loss via aberrant microglia-mediated synaptic elimination.

[0184] Using a photooxidative light damage model of photoreceptor degeneration, evidence of C1q deposition on photoreceptor synapses and microglial phagocytosis of C1q-tagged synapses is provided herein. A significant correlation is shown between the levels of C1q and cell body loss in the retina and photoreceptor synapses. Furthermore, externalization of the C1q substrate, phosphatidylserine (PS), is shown on photoreceptor synapses, suggesting the potential involvement of PS in C1q recruitment onto synapses. And finally, intravitreal treatment with anti-C1q antibodies reduced the levels of complement components in the light-damaged retina (confirming target engagement and neuroprotection after treatment).

[0185] Increased levels of classical complement component C1q were confirmed in retinal lysates from both untreated and IgG Rd10 animals compared to WT (Figure 14B). Anti-C1q treatment resulted in reduced C1q levels in retinal lysates compared to IgG1-treated and untreated rd10 groups (Figure 14B), confirming good measurable PK and C1q involvement in the retina. Photoreceptor synapses (BNS markers) were preserved upon treatment with C1q inhibitors (Figure 15B).

[0186] The present disclosure relates generally to compositions and methods for preventing, reducing the risk of developing, or treating inherited retinal diseases (IRDs) (e.g., retinitis pigmentosa, choroideremia, Stargardt disease, cone-rod dystrophy, and Leber congenital amaurosis) or retinal detachment in human patients.

[0187] Inherited retinal diseases (IRDs) are a group of diseases that can cause severe vision loss or even blindness. Each IRD is caused by at least one gene not functioning properly. IRDs affect individuals of all ages, can progress at different rates, and are rare. However, many are progressive, which means that the symptoms of the disease worsen over time.

[0188] There are many types of IRDs, and others yet to be discovered. The most common types of IRDs include retinitis pigmentosa / rod-cone dystrophy, choroideremia, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis, X-linked RP, and Usher syndrome. The common pathway is photoreceptor degeneration.

[0189] Retinitis pigmentosa (RP) / rod-cone dystrophies are a group of related eye diseases caused by variants in 60 genes that affect the retina. In people with RP, vision loss occurs as the light-sensing cells in the retina gradually disappear. The severity and speed of disease progression can vary in people with RP, depending on the genes affected. RP may first appear in childhood (early-onset RP) or adulthood. The first sign of RP is usually a loss of night vision, called night blindness. RP then causes blind spots to develop in the peripheral (side) vision. Over time, these blind spots progress to reduced peripheral vision. The disease progresses over time, eventually affecting the central vision needed for tasks such as reading, driving, and facial recognition, also known as tunnel vision. Choroideremia is a condition with progressive vision loss that primarily affects males. The first symptom of this condition is usually night blindness, which can occur in early childhood. Over time, a person develops tunnel vision and loses the ability to see details. These vision problems result from the ongoing loss of cells in the retina and nearby blood vessel network (called the choroid). The vision loss in choroideremia worsens over time, but the rate of deterioration varies among affected individuals. The condition can cause complete vision loss by late adulthood.

[0190] Stargardt disease is also called Stargardt macular dystrophy. The disease causes damage to the macula, a small area in the center of the retina, resulting in sharp, straight vision. The disease typically causes central vision loss during childhood or adolescence. Sometimes, vision loss may not be noticed until later in adulthood. People with the disease rarely lose all their vision.

[0191] Cone-rod dystrophies (CRDs) are a group of over 30 IRDs that affect the cones and rods. Cones and rods are light-sensitive cells found in the retina. Due to the gradual deterioration of the cones and rods, people with the condition experience vision loss over time. The first symptoms usually occur during childhood and may include blurred vision and intense sensitivity to light (called photophobia). These symptoms are followed by a blind spot in the center of vision, inability to see color, and loss of side or peripheral vision. Most individuals with the condition lose a significant amount of vision by mid-adulthood.

[0192] Leber congenital amaurosis (LCA) is an eye disorder that primarily affects the retina. The retina is the layer of the eye that functions like the film in a camera, capturing visual images and sending electrical signals to the brain. LCA is one of the earliest onset forms of IRD. People with this disorder typically have severe vision problems that begin in infancy. LCA is also associated with other vision problems such as photophobia: increased sensitivity to light; nystagmus: uncontrollable eye movements; extreme hyperopia: inability to see objects clearly up close, such as book bindings or clock faces; delayed pupillary response: the pupils of individuals with LCA do not respond normally to light, instead, they may open and close more slowly than normal or not respond to light at all; malformed corneas: the cornea is cone-shaped and may be abnormally thin with LCA; crossed eyes (strabismus): the eyes look in two different places at the same time because the eye muscles do not form or function properly.

[0193] X-linked RP (XLRP) is a severe form of retinitis pigmentosa (RP). XLRP is associated with mutations in a gene located on the X chromosome, meaning that the condition primarily affects males. However, some female carriers can also be clinically affected, but usually have a much more severe phenotype than males. The phenotypic variation among female carriers is due to a pattern of random inactivation of the X chromosome carrying the wild-type gene during development of retinal tissue, regulated by other genetic and environmental factors. XLRP is most commonly caused by mutations in the retinitis pigmentosa GTPase (RPGR) gene on the X chromosome. It is characterized by early onset and rapid progression of vision loss, resulting in legal blindness by the end of one's twenties. Less frequent forms of XLRP are caused by mutations in the RP2 and OFD1 genes. Mutations in the RPGR gene can be associated with rod-cone or cone-rod dystrophy phenotypes. Most cases present with a rod-cone dystrophic type of disease progression, with central vision initially being reduced more than peripheral field loss. However, some also have early cone involvement during the early stages of the disease, with corresponding impairment of central vision. The fovea is eventually affected in all cases during the later stages of the disease, with subsequent cone photoreceptor degeneration.

[0194] Usher syndrome, also known as Hallgren syndrome, Usher-Hallgren syndrome, retinitis pigmentosa syndrome, or retinal anomaly syndrome, is a rare genetic disorder caused by a mutation in any one of at least 11 genes that results in a combination of hearing loss and visual impairment. It is a leading cause of blindness and is currently incurable. It causes deafness or hearing loss and an eye disease called retinitis pigmentosa (RP). Sometimes it also causes balance problems. Usher syndrome is classified into three subtypes (I, II, and III) depending on the causative gene and the onset of hearing loss. All three subtypes are caused by mutations in genes involved in the function of the inner ear and retina. These mutations are inherited in an autosomal recessive pattern.

[0195] Retinal detachment refers to a situation in which a thin layer of neural tissue (the retina) at the back of the eye pulls away from its normal position. Retinal detachment separates the retinal photoreceptor cells from the retinal pigment epithelium (RPE), which provides oxygen and nutrients and removes waste products. At this point, the retina is separated from the RPE, and there is photoreceptor degeneration and resultant vision loss. The longer the central retina is detached and the retinal detachment goes untreated, the higher the risk of permanent vision loss in the affected eye. Warning signs of retinal detachment may include the sudden appearance of floaters and blinks, as well as reduced vision. Retinal detachment occurs in approximately 10-12 cases per 100,000 cases per year. In approximately 50% of cases, the central retina detaches. When the central retina detaches, vision recovery only reaches approximately 50% of pre-detachment vision, despite successful reattachment of the retina. The cause of this limited vision recovery is photoreceptor degeneration.

[0196] Genetic or pharmacological inhibition of the classical complement pathway resulted in increased photoreceptor cell survival and improved retinal function. However, the mechanisms by which C1q and the classical complement pathway promote photoreceptor cell degeneration are not fully understood. Based on the described role of C1q in microglia-mediated synapse pruning in CNS development and disease, we hypothesize three mechanisms of damage to photoreceptors mediated by C1q: (1) C1q tags photoreceptor synapses in retinal degenerative disorders, contributing to neuronal loss via abnormal microglia-mediated synapse elimination; (2) C1q is activated by waste products of damaged photoreceptors (phosphatidylserine, c-reactive protein, and altered photoreceptor membranes), leading to damage from recruitment of phagocytes that also bring further C1q; and (3) formation of the membrane attack complex (MAC), which causes activation of the entire classical complement pathway and cell lysis.

[0197] C1q recognizes certain pathogens, denaturation of self-antigens, antigen-binding antibodies or specific molecules on the cell surface. In normal aging, C1q accumulates on synapses, but this weakens, possibly due to age or stress on neuronal cells, and subsequently various pathophysiological stimuli can trigger activation of the classical complement cascade, leading to inappropriate elimination of synapses. This abnormal inflammatory response with synapse elimination is called complement-mediated neurodegeneration (CMND). CMND is involved in Alzheimer's disease, schizophrenia, Huntington's disease, frontotemporal dementia, spinal muscular atrophy and glaucoma. In the presence of degenerative stress in the retina, C1q activation leads to synapse elimination, contributing to RGC and optic nerve loss.

[0198] Using a photooxidative light damage model of photoreceptor degeneration, we provide evidence of C1q deposition on photoreceptor synapses and demonstrate microglial phagocytosis of C1q-tagged synapses. We show a striking correlation between the levels of C1q and cell body loss in the retina and photoreceptor synapses. We also show externalization of the C1q substrate, phosphatidylserine (PS), on photoreceptor synapses, suggesting a potential involvement of PS in C1q recruitment onto synapses. And finally, we show that intravitreal treatment with anti-C1q antibodies reduces the levels of complement components in light-damaged retinas. Additionally, we confirm C1q deposition on photoreceptor synapses in human retinal tissue map atrophy, suggesting that this mechanism is relevant to humans.

[0199] The present disclosure relates generally to compositions and methods for preventing, reducing the risk of developing, or treating inherited retinal diseases (IRDs) (e.g., retinitis pigmentosa, choroideremia, Stargardt disease, cone-rod dystrophy, and Leber congenital amaurosis) or retinal detachment in human patients.

[0200] Such methods include administering to the patient via intravitreal injection a composition comprising about 1 mg to about 10 mg of anti-C1q antibody (e.g., about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg or about 10 mg of anti-C1q antibody). Such a method also includes administering to a patient via intravitreal injection a composition comprising about 1 mg to about 10 mg (e.g., about 1 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3 mg, about 3.5 mg, about 4 mg, about 4.5 mg, about 5 mg, about 5.5 mg, about 6 mg, about 6.5 mg, about 7 mg, about 7.5 mg, about 8 mg, about 8.5 mg, about 9 mg, about 9.5 mg, or about 10 mg of anti-C1q antibody), 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, and 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 composition administered may comprise about 1 mg to about 5 mg of anti-C1q antibody. The composition administered may comprise about 1 mg to about 2.5 mg, about 2.5 mg to about 5 mg, about 5 mg to about 7.5 mg, or about 7.5 mg to about 10 mg of anti-C1q antibody. The composition administered may comprise about 5 mg of anti-C1q antibody. The composition administered may comprise about 10 mg of anti-C1q antibody. In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, the 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 light chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 4 and 35-38.In some embodiments, the antibody comprises a heavy chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 8 and 31-34, the 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. In some embodiments, the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 8 and 31-34. In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% homology with an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, the 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, and a heavy chain variable domain comprising an amino acid sequence having at least about 95% homology with an amino acid sequence selected from SEQ ID NOs: 8 and 31-34, the 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. In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, and a heavy chain variable domain comprising an amino acid sequence selected from SEQ ID NOs: 8 and 31-34. In some embodiments, the antibody may be a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, an antibody fragment, or an antibody derivative thereof. The antibody fragment may be 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 Fab fragment comprises a heavy chain Fab fragment of SEQ ID NO:39 and a light chain Fab fragment of SEQ ID NO:40.

[0201] In some embodiments, the antibody is administered once a week, once every two weeks, once every three weeks, once a month, once every four weeks, once every six weeks, once every eight weeks, once every two months, once every ten weeks, once every twelve weeks, once every three months, or once every four months. In some embodiments, the antibody is administered for at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

[0202] In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 2.5 mg / eye once every month, once every four weeks, once every six weeks, or once every other month.

[0203] In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 5mg / eye once a month, once every 4 weeks, once every 6 weeks, or once every two months.In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 5mg / eye once a month or once every 4 weeks.In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 5mg / eye once every 6 weeks.In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 5mg / eye once every two months or once every 8 weeks.

[0204] In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 10mg / eye once a month, once every 4 weeks, once every 6 weeks, or once every two months.In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 10mg / eye once a month or once every 4 weeks.In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 10mg / eye once a month or once every 6 weeks.In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 10mg / eye once a month or once every 8 weeks.

[0205] FabA injections are completed using sterile technique by physicians trained and certified to perform IVT injections.

[0206] Anti-C1q antibodies may inhibit interactions between C1q and autoantibodies, or between C1q and C1r, or between C1q and C1s, or may promote the clearance of C1q from the circulation or tissues. In some embodiments, anti-C1q antibodies have a dissociation constant (K D In some embodiments, the anti-C1q antibody binds to C1q with a binding stoichiometry ranging from 20:1 to 1.0:1 or less than 1.0:1, a binding stoichiometry ranging from 6:1 to 1.0:1 or less than 1.0:1, or a binding stoichiometry ranging from 2.5:1 to 1.0:1 or less than 1.0:1.

[0207] The method inhibits a biological activity of C1q, such as (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 characterized by: (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 red blood cell lysis; (6) reduced erythrophagocytosis; (7) reduced dendritic cell infiltration; (8) inhibition of complement mediated red blood cell 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 erythrocyte infiltration; (18) reduced erythrocyte lysis; (19) reduced lymphocyte infiltration; (20) reduced macrophage infiltration; (21) reduced antibody deposition; (22) reduced erythrocyte lysis; (23) reduced erythrocyte lysis; (24) reduced erythrocyte lysis; (25) reduced erythrocyte lysis; (26) reduced erythrocyte lysis; (27) reduced erythrocyte lysis; (28) reduced erythrocyte lysis; (29) reduced erythrocyte infiltration; (30) reduced erythrocyte lysis; (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 Decreased neutrophil infiltration, (13) decreased platelet phagocytosis, (14) decreased platelet lysis, (15) improved graft survival, (16) decreased macrophage-mediated phagocytosis, (17) decreased autoantibody-mediated complement activation, (18) decreased red blood cell destruction in transfusion reactions, (19) decreased alloantibody-induced red blood cell lysis, (20) decreased hemolysis in transfusion reactions, (21) decreased alloantibody-mediated platelet lysis, (22) improved anemia, (23) decreased eosinophilia, (24 ) reduced C3 deposition on red blood cells (e.g., reduced C3b, iC3b, etc. deposition on RBCs), (25) reduced C3 deposition on platelets (e.g., reduced C3b, iC3b, etc. deposition on platelets), (26) reduced anaphylatoxin production, (27) reduced autoantibody-mediated rash formation, (28) reduced autoantibody-induced lupus erythematosus, (29) reduced red blood cell destruction in transfusion reactions, (30) reduced platelet lysis in transfusion reactions, (31) obesity These include decreased cellular 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.

[0208] In some embodiments, the CH50 hemolysis comprises human CH50 hemolysis. The antibody may be capable of neutralizing at least about 50% to about 100% of human CH50 hemolysis. The antibody may be capable of neutralizing about 50%, about 60%, about 70%, about 80%, about 90%, about 100% of human CH50 hemolysis. The antibody may 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.

[0209] In some embodiments, the antibody is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a human antibody, a chimeric antibody, a monovalent antibody, a multispecific antibody, or an antibody fragment, 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. Examples of antibody fragments are Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, and single chain antibody molecules.

[0210] It is contemplated that the compositions may be obtained and used under the guidance of a physician for in vivo use. The dosage of the therapeutic formulation may vary widely depending on the nature of the disease, the frequency of administration, the mode of administration, clearance of the drug from the host, and the like.

[0211] As used herein, "chronically administered," "chronic treatment," "chronically treating," or similar grammatical variants thereof, refers to a treatment regimen used to maintain a predetermined threshold concentration of a therapeutic agent in the patient's eye to completely or substantially suppress systemic complement activity in the patient over an extended period of time. Thus, a patient who is chronically treated with an anti-C1q antibody may be treated with an anti-C1q antibody for 2 weeks or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks; 1, 2, 3 or 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 12 years, or for the remainder of the patient's life). In some embodiments, the antibody may be administered chronically to a patient in need thereof in an amount and frequency effective to maintain serum hemolytic activity at 20% or less (e.g., less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or even less than 5%). In some embodiments, the antibody may be administered to a patient in an amount and frequency effective to maintain serum lactate dehydrogenase (LDH) levels within at least 20% of the normal range for LDH (e.g., less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or even 5%).

[0212] Therapeutic agents, such as anti-C1q antibodies, can be incorporated into a variety of formulations for therapeutic administration by combining with an appropriate, pharma- ceutically acceptable carrier or diluent. EXAMPLES

[0213] Example 1: Methods Photooxidative white light damage model Male Balb / C mice (12 weeks old, Charles River Laboratories) were used in the studies described in Examples 2-7. Animals were dark-adapted overnight for the experiments with a single extension of the dark phase of 3-4 hours before the onset of light induction (acute: 25,000 lux for 4 hours, mild: 5000 lux for 30 minutes). Acute light settings were used for model characterization (Results Figure 1). Mild light settings were used for all other experiments (Results Figures 2-5). Animals were sacrificed and tissues were collected at baseline, day 1, day 3 and day 7.

[0214] Rd10 mouse model Rd10 mice were dark-reared from birth and transferred to housing in a light-controlled environment (normal circulating daytime light ∼200 lux) on P30-P31.

[0215] Anti-C1q antibody treatment (or IgG control antibody) was administered intraperitoneally (ip) at a dose of 100 mg / kg twice weekly starting on P12. For ip injections, mice were gently restrained by hand and an 8 mm 31-gauge needle was used to puncture the abdomen to deliver the antibody. Animals were sacrificed and tissues were collected at P30, P33, and P38.

[0216] Terminal plasma and ocular tissue collection Animals were anesthetized and approximately 300-500 μl of terminal whole blood was collected via cardiac puncture. Blood was immediately placed into K2EDTA tubes and centrifuged at 5,000 x g for 10 min to obtain plasma. Plasma was transferred to 1.7 mL snap-cap tubes and stored at -80 °C until shipment to the client on dry ice. For biochemical evaluation, animals were perfused transcardially with saline and eyes were enucleated. Retinas / RPE were isolated, weighed, snap frozen, and stored at -80 °C. For histopathological evaluation, animals were perfused transcardially with saline followed by 4% PFA (minimum 10 min / 50 ml). After saline / PFA perfusion, eyes were collected. Posterior eye cups were fixed in 4% PFA for 2 h, transferred through a serial sucrose gradient (10%, 20%, 30%), embedded in optimal cutting temperature compound (OCT), and frozen.

[0217] Intravitreal injection Animals were sedated with isoflurane. Under an operating microscope, eyes were inserted into the mid-vitreous with a 32G needle. For PSVue labeling, eyes were injected with 2 μl of 1 mM PSVue 6 hours prior to tissue collection. The contralateral eye was used as a non-injected control. For anti-C1q treatment, 1 μl of optimized mouse anti-C1q antibody (Mab3, optimized from Mab1) or IgG1 MOPC control antibody (7.5 μg / μl) was injected bilaterally 1 day prior to light exposure, and tissue collection was performed at baseline, day 3, and day 5.

[0218] ELISA Animals were perfused transcardially with saline and eyes were enucleated. Retinas / RPE were isolated, weighed, snap frozen and stored at -80°C. Frozen tissues were homogenized using a pestle moto in lysis buffer (25 mM Hepes, 0.1 M NaCl, 1% Triton X100, complete protease inhibitor cocktail; or 10 mM EDTA and Thermo Scientific #A32965 in Tris-buffered saline). Proteins were quantified using a Micro BCA Assay Kit (Thermo Scientific #23235). Complement signatures were determined using a standard sandwich ELISA assay. Briefly, black 96-well ELISA plates (Costar #3925) were coated overnight at 4°C with 10 μg / mL capture antibody in 75 μL of bicarbonate buffer (pH 9.4). The next day, the plates were washed with dPBS (pH 7.4) and blocked with dPBS buffer containing 3% bovine serum albumin (BSA, VWR #28382). Standards and samples were prepared in assay buffer (dPBS containing 0.3% BSA, 0.1% tween and 0.05% EDTA) and added to the plates (75 μL per well) after removing the blocking solution. The plates were incubated overnight at 4° C. with shaking at 300 rpm. The next day, the plates were washed three times with wash buffer (dPBS containing 0.05% Tween) and incubated with detection antibody (75 μl per well) for 1 hour at room temperature with shaking. After incubation, the plates were washed three times with wash buffer developed by adding alkaline phosphatase substrate (Life Technologies, T2214) and read using a luminometer. For the PK assay, mouse C1q (M099, Complement Tech) and AP-conjugated anti-mouse antibody (115-055-071, Jackson ImmunoResearch) were used as capture and detection antibodies, respectively. For the C1q assay, a house capture antibody (JL1 clone, Atum) and detection antibody (M1-AP, antibody solution 1:3000) were used.C1s assay was performed using anti-mouse C1s (LSBio, 2ug / ml) and anti-mouse C1s-AP (LSBio, 1:5000) as capture and detection antibodies, respectively. C3d assay was performed using anti-human C3d (Dako, 1ug / ml) and anti-mouse C3 (11H9 clone, Abcam, 1:1000) as capture and detection antibodies, respectively. Albumin assay was performed using a kit (Abcam, ab108792). Standards were fitted using 4PL logistic fit and unknowns were converted to concentrations, corrected for dilution, and then plotted using GraphPad Prism.

[0219] immunohistochemistry Animals were perfused transcardially with saline followed by 4% PFA (minimum 10 min / 50 ml). After saline / PFA perfusion, eyes were collected. The posterior eye cups were fixed in 4% PFA for 2 h, transferred through a successive sucrose gradient (10%, 20%, 30%), embedded in optimal cutting temperature compound (OCT), and frozen. After embedding, 10 μm thick sections were cut using a cryostat, collected serially on microscope slides (Superfrost plus, VWR), and stored at -80 °C until further use. For histological staining, retinal sections were washed in PBS, blocked in blocking buffer (PBS containing 4% donkey serum, 0.3% Triton X-100) for 1 h, and incubated with primary antibodies overnight at 4 °C. The next day, sections were washed and incubated for 2 h with appropriate Alexa-fluorophore conjugated secondary antibodies (ThermoFisher Scientific), washed and coverslipped with Fluoromount G (Southern Biotech). All washing steps were 3 × 10 min in PBS. Nuclei were counterstained with Dapi. The following antibodies were used: in-house rabbit anti-C1q (clone 4.8, ATUM, 1:500), mouse anti-bassoon (Abcam, 1:500), guinea pig anti-vGlut1 (Millipore Sigma, 1:500), chicken anti-homer1 (Synaptic System, 1:500), goat anti-Iba1 (Novus, 1:500), rat anti-CD68 (BioRad, 1:200). Rabbit IgG isotype antibody (ThermoFisher) was used as a negative control for C1q staining. Images were captured at 10x or 20x magnification using an epifluorescence microscope (Leica) and at 63x magnification using confocal microscopy (Leica, Stellaris 5). Images were processed using ImageJ.

[0220] C1q synaptic colocalization and phagocytosis analysis Two z-stack images were imaged per section (z-stack size: 0.7um, 12 planes) and at least three sections per animal were analyzed. 3D volumetric surface renderings of each z-stack were created (Imaris Software). The retinal OPL was selected as the region of interest. Surface-rendered images were used to identify microglial surface and volume, as well as synaptic and C1q elements. C1q-tagged synapses were quantified based on proximity (C1q and bassoon elements at distance ≤ 400μm). Engulfed synapses were quantified based on overlap (bassoon-Iba1 overlap ratio ≥ 70%) and calculated as the number of engulfed C1q-tagged synapses / total number of synapses.

[0221] Phosphatidylserine (PS)-C1q binding assay Phosphatidylserine (PS) and phosphatidylcholine (PC) lipid microparticles were purchased from Echelon Biosciences (numbers P-B1PS and P-B1PC). Purified human C1q was purchased from Complement Technologies (number A099). Ninety-six (96) well clear round bottom plates were used for the assay (VWR number 353227). Microparticle suspensions were prepared according to the manufacturer's instructions. Briefly, the microparticles were first thoroughly vortexed to ensure a homogenous bead suspension. Washing, dilution, titration, and incubation steps were performed in Annexin V binding buffer (Thermo Fisher number BMS500BB). All washing steps were performed at a centrifugation speed of 10,000 g for 10 minutes. Beads were diluted 20-fold before use. For each test point, 150 μL of bead suspension (approximately 100,000 beads) was used. Microparticles were washed twice and resuspended in binding buffer. Fifty microliters per well was considered as the reaction volume. C1q titrations (1:10 dilution, 100-0.1 μg / mL) were prepared in binding buffer. C1q and lipid microparticles were added to the wells in a 1:1 ratio and incubated at 37°C for 30 minutes. After incubation, the microparticles were washed twice with one volume of flow buffer (PBS, 1% BSA, 2 mM EDTA). The supernatant was decanted and the microparticles were resuspended in flow buffer (100ul per well) containing anti-C1q-APC (Dako, 1:1000) and incubated at 4°C for 30 minutes. After incubation, the microparticles were washed twice, resuspended in flow buffer (150μl) and analyzed via flow cytometry utilizing the green channel for microparticles (FITC) and the far-red channel for C1q binding (APC).

[0222] Complement deposition assay Washing, dilution, titration, and incubation steps were performed in GVB++ buffer (Complement Technology number B100). PS and PC lipid microparticles were washed twice and resuspended in the appropriate volume, allowing for approximately 100,000 beads for each test point. Human serum titrations were prepared in GVB++. Ninety-six (96) well clear round bottom plates were used for the assay (VWR 353227). GVB EDTA (Complement Technologies number B105) was used as a negative control buffer. The reaction mixture contained a 1:1:1 ratio of buffer (GVB++ or GVB EDTA, + / - titrated anti-C1q antibody):lipid microparticle:human serum in a final volume of 33.3 μl. After 30 min incubation at 37° C., the plate was washed twice with GVB++ (150 μl). Microparticles were resuspended in flow buffer (PBS, 1% BSA, 2 mM EDTA) containing anti-C1q-APC (Dako, 1:2000) or anti-C4-APC (Dako, 1:500) and incubated for 30 min at 4° C. After incubation, microparticles were washed twice, resuspended in flow buffer (150 μl) and analyzed via flow cytometry using the green channel for microparticles (FITC) and the far-red channel for C1q or C4 deposition (APC).

[0223] Human GA donor tissue procurement and immunohistochemistry Human donor eyes were obtained within 24 hours postmortem from the San Diego Eye Bank (California, USA). Clinical records and family questionnaires of all donors were obtained. Human eyes were fixed in 4% for 2 hours than transferred to PBS overnight. The following day, the posterior eye cups were cryoprotected in sucrose 30% for 24–48 hours. The temporal, nasal, superior and inferior regions were then sampled using a 6 mm diameter dissecting trephine (Biomedical Research Instruments, MD, USA) using the same trephine that isolated the macula. Retinal samples were embedded in OCT and frozen. After embedding, 10 μm thick sections were cut using a cryostat and collected serially on microscope slides (Superfrost plus, VWR) and stored at −80°C until further use. Histological staining of human donor retinal sections was performed as described above for mouse samples. The following antibodies were used: rabbit anti-human C1q (Dako, 1:500), guinea pig anti-vGlut1 (Millipore Sigma, 1:500), chicken anti-homer1 (Synaptic System, 1:500). Rabbit IgG isotype antibody (ThermoFisher) was used as a negative control for C1q staining. Images were captured at 10x magnification using an epifluorescence microscope (Leica) and at 63x magnification using confocal microscopy (Leica, Stellaris 5). Images were processed using ImageJ.

[0224] statistical analysis All data are presented as mean ± standard deviation (SD). Statistical analysis was performed with GraphPad Prism using unpaired Student's t-test, or one-way analysis of variance (ANOVA) or two-way analysis of variance, followed by Bonferroni or Dunnet or Sidak or Tukey post-hoc tests. Data are presented as mean ± SD. All p-values ​​are indicated in the figure legends and were considered statistically significant when less than 0.05.

[0225] Example 2: Loss of photoreceptor synapses and increased microgliosis in a photooxidative light damage model Presynaptic marker Bassoon was used to identify synapses, and epifluorescence was used to measure synapse density. The number of photoreceptor nuclear columns was used as a measure of photoreceptor survival. Iba1, a calcium-binding protein specifically expressed in microglia, was used as a pan-microglia / macrophage marker. CD68, a lysosomal protein, was used to identify reactive phagocytic microglia. Progressive photoreceptor synapse and cell body loss (Figures 1A-1C), as well as increased microglial reactivity (Figures 1D-1F), were observed after photooxidative injury. Notably, the distribution of phagocytic microglia in the synaptic layer peaked at day 1, the same time point at which significant synapse loss was first observed (Figure 1E).

[0226] Example 3: Increased C1q levels on photoreceptor synapses correlate with photoreceptor synapse loss in a photooxidative light damage model of photoreceptor degeneration Increased levels of the initiating classical complement components C1q and C1s, as well as the downstream activation product C3d, were observed in retinal lysates from light-exposed animals by standard ELISA (Figures 2A-2C). We next investigated retinal C1q distribution by immunofluorescence. As shown in Figure 2D, C1q (i) colocalized with Iba1 (iii) and Bassoon (iv), confirming C1q expression in microglia / macrophages and expression / deposition on synapses. A significant negative correlation was observed between C1q expression and photoreceptor synapse density (Figure 2G, Pearson r = -0.72, p = 0.00003), suggesting a causal relationship (Figure 2E).

[0227] Example 4: Microglial phagocytosis of photoreceptor C1q-tagged presynaptic elements following photooxidative damage To assess microglial phagocytosis of C1q-tagged synapses, we triple-labeled retinal sections from naïve and light-exposed mice for C1q, Bassoon, and Iba1 (Figure 3A). High-resolution imaging was performed using confocal microscopy. Phagocytosis analysis was performed using 3D reconstruction and surface rendering software (Figure 3B). A marked reduction in synaptic density (Figure 3C) was associated with a significant increase in the percentage of C1q-tagged synapses in light-damaged retinas compared to naïve (Figure 3D) and a significant increase in microglial phagocytosed C1q-tagged synapses (Figure 3E).

[0228] Example 5: Phosphatidylserine binds C1q and is externalized onto photoreceptor synapses after photooxidative light damage To test whether PS exposure occurs at photoreceptor synapses during disease, we performed IVT administration of the PS-binding probe PSVue (PSVue 550) in a light damage model (Scott-Hewitt N et al, The EMBO Journal, 2020). Retinal tissue was processed and analyzed using IHC 3 days after light exposure. Increased PSVue labeling was detectable in the OPL and ONL of light-damaged retinas compared to naïve (Figure 4A). High-resolution confocal imaging and 3D reconstruction / surface rendering confirmed the colocalization of PSVue labeling with C1q and Bassoon (Figure 4A). Notably, PSVue-positive surfaces appeared in close proximity to both Bassoon- and C1q-positive surfaces in a sandwich fashion (Figure 4A iii), indicating PS externalization and C1q interaction on synapses.

[0229] To assess whether PS directly binds C1q and activates the complement cascade, C1q binding and complement deposition assays were performed using PS-lipid-green-fluorescent-microparticles. Phosphatidylcholine (PC)-lipid-green-fluorescent microparticles were used as a negative control. Direct binding of C1q to PS lipid microparticles, but not to PC, was observed after incubation with C1q at a concentration of 10 ng / mL (Figure 4B). Deposition of both C1q and C4 was observed on PS lipid microparticles, but not on PC lipid microparticles. Little or no deposition was observed in the negative control group (Figures 4C and D). Titration of anti-C1q neutralizing antibody (Mab1-Fab) in serum resulted in reduced deposition of C1q and C4 on PS lipid microparticles (Figures 4E and 4F), suggesting that competitive binding of anti-C1q antibodies reduced PS-mediated complement activation.

[0230] Example 6: Intravitreal anti-C1q treatment reduced retinal complement component levels following photooxidative light damage Intravitreal administration of a neutralizing optimized murine anti-C1q antibody (Mab3, optimized from Mab1) was performed 1 day prior to exposure to light injury. Detectable drug levels and selected complement component levels were assessed by standard ELISA 3 days after light injury. Measurable drug levels were found in retinal lysates from animals receiving anti-C1q treatment, but not in IgG1-treated or untreated groups (Figure 5A). Increased levels of classical complement components C1q and C1s, as well as downstream activation component C3d, were confirmed in retinal lysates from both untreated and IgG-treated light-exposed animals compared to naïve (Figure 5B-5D). Significant decreases in C1q, C1s, and C3d were observed following anti-C1q treatment compared to IgG antibody (Figure 5B-5D). Validation studies confirm target engagement and evaluate neuroprotection following treatment.

[0231] Example 7: C1q expression and deposition on photoreceptor synapses in human GA donor retinas GA donor eyes were procured and subjected to histological assessment of C1q distribution across retinal layers. Synaptic integrity was assessed by immunofluorescence in the macular region of GA donors versus healthy donor retinas. Reduced immunoreactivity of the presynaptic marker Vglut1 and increased labeling of C1q in the photoreceptor synaptic layer confirmed the synaptic loss and accumulation of C1q occurring in GA retinas compared to healthy donors (Figure 6A-6B). Finally, triple immunolabeling of C1q (grey), presynaptic maker Vglut1 and postsynaptic marker (Homer1) confirmed colocalization of C1q with photoreceptor synapses in human GA donor retinas (Figure 6C).

[0232] Example 8: Evaluation of FabA in non-clinical trials FabA drug products are sterile isotonic solutions for IVT injection.

[0233] FabA is provided as a sterile single-dose vial for IVT injection.

[0234] FabA was used in an extensive series of in vitro and in vivo pharmacological studies.

[0235] The antibodies Mab1, Mab1-Fab, and Mab2 were active in an acute mouse model of glaucoma, protecting retinal ganglion cells and / or nerve fibers from loss. In a mouse model of photooxidative light-induced damage, intravitreally administered Mab1 protected against photoreceptor cell loss and functional retinal connectivity in the eye.

[0236] GLP studies of FabA consisted of a single-dose rat ocular toxicity study, and three repeat-dose cynomolgus monkey ocular toxicity studies. The route of administration for the toxicity studies was IVT injection. In single-dose and two-dose (once monthly) IVT GLP studies, FabA has shown no evidence of adverse ocular toxicity at a no observed adverse effect level (NOAEL) of 5 mg / eye (equivalent to a 10 mg human dose) once monthly in cynomolgus monkeys, and 0.05 mg / eye (equivalent to a 10 mg human dose) in single-dose rat studies. A 26-week chronic ocular toxicity study in cynomolgus monkeys determined that adverse ocular changes were associated with the two-injection procedure and / or anti-drug antibody (ADA) mediated, and not a direct effect of FabA IVT administration.

[0237] Pharmacokinetic evaluation of FabA in serum and vitreous of rats and cynomolgus monkeys was performed. Vitreous C1q concentrations were measured as a PD marker of C1q inhibition of FabA in monkeys. PK / PD and TK / PD studies in monkeys demonstrated robust ocular PD effects consistent with FabA drug exposure levels in the vitreous.

[0238] Binding and affinity of FabA and precursor molecules to human C1q The binding affinity of FabA and precursor molecules to C1q was also investigated by ELISA. All molecules (Mab2-Fab, FabA, and Mab2, Mab1, and Mab1-Fab) showed affinity for human C1q with 50% effective concentrations (EC50) ranging from 2.2 to 4.9 ng / mL (20 to 95 pM). The EC50 for FabA binding to C1q is 2.5 ng / mL.

[0239] Effect on IgM-mediated erythrocyte hemolysis The ability of FabA, Mab2, and Mab2-Fab to functionally inhibit classical complement-dependent hemolysis of IgM-opsonized RBCs in human serum was measured (Figure 9). The three molecules showed nearly identical potencies, consistent with comparable binding affinities. The 50% inhibitory concentration (IC50) of FabA inhibition of IgM-coated RBC hemolysis is 0.62 μg / mL (approximately 12 nM).

[0240] In vivo pharmacology studies Anti-C1q antibody treatment prevents optic nerve damage in a mouse model of acute glaucoma In mice, injection of polystyrene beads into the anterior chamber induces a sudden rise in IOP, loss of retinal ganglion cells, and optic nerve damage over a period of 2 weeks. Mab1, Mab1-Fab, and Mab2 were administered intravitreally to mice the day before and 7 days after IOP elevation. 2 μL of 10 mg / mL antibody or saline was administered at each time point. Based on a vitreous volume of 5-10 μL in mouse eyes, antibody concentrations were 2000-4000 μg / mL. Optic nerves were harvested 2 weeks after injury, and the number of intact and damaged axons was quantified. Anti-C1q antibody treatment provided protection against RGC loss and / or retinal nerve fiber damage in this induced mouse model of glaucoma (Figure 10).

[0241] Anti-C1q antibody treatment protects photoreceptor cells from damage in a photooxidative light-induced injury model. When mice were exposed to 100,000 lux of natural white LED for 1-7 days, photooxidative damage resulted in retinal photoreceptor loss. In this model, there was a time-dependent increase in C1qa gene expression over 3-7 days that correlated with photoreceptor cell death and microglia / macrophage recruitment. C1qa- / - mice showed less photoreceptor cell death, reduced microglia / macrophage recruitment to photoreceptor cell injury, and higher visual function at 14 days, but not at 7 days, after light damage induction. IVT administration of Mab1 antibody 7 days after light injury reduced photoreceptor cell loss and preserved retinal function as measured by electroretinogram (Figure 11). Mice were administered 1 μL of 7.5 mg / mL antibody, which corresponds to a concentration of 750-1500 ug / mL in the vitreous. In contrast, systemic delivery of Mab1 at 100 mg / kg on days 0, 4, and 8 had no effect on photoreceptor loss or function. Retinal C1q was expressed primarily by the outer retina in early AMD and subretinal microglia / macrophages located in the mouse retina. Thus, protection by anti-C1q antibodies suggests a distinct role for C1q in photoreceptor damage and initiation of the classical complement cascade in the pathogenesis of the human disease GA.

[0242] Safety Pharmacology Systemic exposure following chronic IVT administration in cynomolgus monkeys for 26 weeks did not exceed 86.3 ng / mL, whereas systemic exposure of Mab2 with the same CDR administered IV once weekly in a 26-week cynomolgus monkey study exceeded 1 mg / mL at a NOAEL of 200 mg / kg.

[0243] Thus, safety pharmacology endpoints of the full length antibody, Mab2, following IV administration up to 200 mg / kg weekly in a 4-week repeat-dose GLP toxicity study in monkeys and up to 200 mg / kg weekly in a 26-week repeat-dose toxicity study in monkeys, with no evidence of treatment-related effects on cardiovascular, respiratory, or neurological endpoints, supporting the systemic safety of IVT-administered FabA.

[0244] Additionally, safety pharmacology endpoints of FabA following SC administration up to 20 mg / kg daily in a 4-week repeat-dose GLP toxicity study in monkeys, with no evidence of treatment-related effects on cardiovascular, respiratory, or neurological endpoints, supporting the systemic safety of IVT-administered FabA.

[0245] Pharmacokinetics in animals Nonclinical studies designed to characterize the PK, TK, and PD of FabA have been conducted in rats and cynomolgus monkeys. These studies include single-dose IVT PK studies in rats and cynomolgus monkeys and repeat-dose TK / PD studies in cynomolgus monkeys with FabA. A larger TK / PD study was conducted in monkeys, with no ocular toxicity in either rat or monkey single-dose studies.

[0246] Pharmacokinetic / toxicokinetic / pharmacodynamic analysis Pharmacokinetics of intravitreal FabA Rats received a single dose of FabA via bilateral IVT at doses of 0.01 mg / eye (equivalent to a 2 mg human dose) or 0.05 mg / eye (equivalent to a 10 mg human dose), after which the drug was relatively rapidly cleared from the vitreous at both dose levels, consistent with a half-life of approximately 12 hours. Cynomolgus monkeys also received FabA via bilateral IVT. The drug was distributed slowly from the vitreous compared to rats, with a half-life of approximately 3 days in both the 1 mg / eye (equivalent to a 2 mg human dose) and 5 mg / eye (equivalent to a 10 mg human dose) dose groups.

[0247] In both species, the IVT PK of FabA was linear with respect to dose. Data from an ocular toxicity study in cynomolgus monkeys, in which FabA was administered twice over a 28-day period at doses of 1.0 mg / eye (equivalent to a 2 mg human dose), 2.5 mg / eye (equivalent to a 5 mg human dose), or 5.0 mg / eye (equivalent to a 10 mg human dose), indicate that vitreous concentrations at the time of sacrifice (i.e., 15 and 30 days after the second dose) were generally consistent with data from single IVT administration.

[0248] In a 26-week chronic ocular toxicity study in cynomolgus monkeys dosed with FabA IVT at 2.5 mg / eye monthly (equivalent to a 5 mg human dose), 5 mg / eye monthly (equivalent to a 10 mg human dose), or 5 mg / eye every other week (all 5 mg / eye doses were subsequently reduced to 2.5 mg / eye and referred to as 5 / 2.5 mg / eye), vitreous fluid FabA concentrations were quantifiable on day 184 in all animals receiving FabA through day 169, and were below the limit of quantification (BQL) on days 242 / 243 after a 10-week treatment-free recovery period in all animals. Vitreous fluid FabA concentrations showed high variability with no clear differences or trends between treatment groups or between sexes.

[0249] Pharmacokinetics of FabA in serum After a single IVT dose, serum concentrations were much lower than vitreous humor concentrations, and C max Serum / C max The vitreous was approximately 0.003 in rats and 0.000001 in cynomolgus monkeys. Serum concentrations were low and the highest mean peak concentration (C max ) was 10.1 ng / mL, which was observed after a second IVT dose of 5 mg / eye (corresponding to a human dose of 10 mg). As FabA partitions from the vitreous into the serum compartment, it can bind to C1q or remain in free form and quantifiable in the assay, with mean C1q of 3.3 and 10.1 ng / mL in the 1 mg / eye group and 2.5 and 5.0 mg / eye groups, respectively. max , resulting in low FabA serum concentrations.

[0250] In contrast, following IV administration of FabA at a dose of 10 mg / kg, maximum FabA concentrations were 13,800 and 17,000 ng / mL in the two cynomolgus monkeys tested, and concentrations declined very rapidly thereafter, consistent with a half-life of approximately 2 hours, as expected for a Fab fragment.

[0251] When comparing serum FabA concentrations after two bilateral IVT doses (5 mg / eye) over a 28-day period versus systemic IV administration of Mab2 at a dose of 200 mg / kg once weekly for 4 weeks, FabA serum exposure was significantly lower (FabA / Mab2 Cmax ratio of 0.00000701).

[0252] In a 26-week chronic ocular toxicity study in cynomolgus monkeys, FabA was administered IVT at doses of 2.5 mg / eye monthly, 5 / 2.5 mg / eye monthly, or 5 / 2.5 mg / eye bimonthly. FabA systemic exposure in serum was low and consistent with the local route of administration. FabA serum concentrations did not exceed 86.3 ng / mL on day 85, and FabA serum concentrations did not exceed 60.8 ng / mL on day 169 after the last dose. Maximum serum FabA concentrations were observed 24-48 hours after dosing, regardless of dose level / regimen and evaluation date. Serum FabA half-life (T1 / 2) values ​​were calculable / reportable only in a small number of animals in the biweekly 5 / 2.5 mg / eye group, ranging from 49.9 to 143 hours on all evaluation dates, representing the potential for distribution from the ocular cavity to serum. Repeated monthly IVT dosing at 2.5 mg / eye resulted in little accumulation of FabA in serum. However, there were increasingly more calculable FabA serum concentrations in this group on each subsequent evaluation day after Day 1. Accumulation could not be determined from Day 1 in any other group due to changes in dose levels after Day 57. Area under the curve versus time "t" (AUC[0-t]) ratios on Day 169 / Day 85 ranged from 0.0407 to 0.664 for males and females receiving the monthly 5 / 2.5 mg / eye dose and from 0.132 to 7.15 for males and females receiving the biweekly 5 / 2.5 mg / eye dose. When comparing gender-matched mean systemic exposure of FabA and Mab2 after 26 weeks of chronic dosing in cynomolgus monkeys, FabA exposure AUC0-t (1,230 hr*ng / mL or 1.23 hr*μg / mL) after bilateral IVT dosing at 5 / 2.5 mg / eye every other week was significantly lower than the AUC0-t (3,150,000 hr*μg / mL) of 200 mg / kg obtained after weekly systemic IV dosing of Mab2. FabA serum exposure was significantly lower (C of 0.000000073 for FabA / Mab2). max ratio, AUC0-t ratio of 0.00000039).

[0253] Ocular C1q Pharmacodynamics The mean vitreous free C1q concentration in control animals was 40.3 ng / mL, whereas in the vitreous of cynomolgus monkeys receiving a single IVT dose of either 1 mg / eye (corresponding to a human dose of 2 mg) or 5 mg / eye (corresponding to a human dose of 10 mg), free C1q concentrations remained below the detection limit (<1.953 ng / mL) for the duration of the study (30 days), indicating complete C1q suppression. In monkeys receiving FabA every 28 days for a total of two doses, C1q remained suppressed for 15 days after the second dose at all three dose levels (i.e., 1, 2.5, and 5 mg / eye, given twice every 28 days). Thirty days after the second FabA dose, C1q remained below the detection limit in some, but not all, eyes.

[0254] Fifteen days after the second 5 mg / eye IVT dose, >80% of C1q was bound to FabA in the retina, choroid, and optic nerve head. Thirty days after the second 5 mg / eye dose, C1q remained suppressed only in the retina and choroid.

[0255] In a 26-week chronic ocular toxicity study in cynomolgus monkeys, FabA was administered IVT at 2.5 mg / eye monthly, 5 / 2.5 mg / eye monthly, or 5 / 2.5 mg / eye every other week. All groups receiving FabA had reduced vitreous C1q concentrations at sacrifice necropsy. Animals with treatment holidays and / or a 10-week treatment-free recovery period had recovered vitreous C1q concentrations at sacrifice necropsy and recovery necropsy, respectively, that were comparable to controls.

[0256] Inhibition of serum C1q and serum hemolysis Following bilateral IVT administration of 5 mg / eye (corresponding to a human dose of 10 mg) to cynomolgus monkeys, C1q-dependent serum hemolysis was inhibited by approximately 50–80%, sustained for approximately 24–48 h after administration of the first IVT dose and for up to 96 h after administration of the second FabA dose, after which it returned to baseline.

[0257] After a single IV dose of 10 mg / kg to cynomolgus monkeys, maximum inhibition of C1q-dependent serum hemolysis was reached at 1 hour. Maximum inhibition was maintained for approximately 24 hours, returning to baseline 120 hours after FabA administration. Serum free C1q also rapidly decreased but had not returned to baseline values ​​by 120 hours, suggesting that some FabA remained bound to circulating C1q throughout this time frame.

[0258] toxicology The safety of FabA is supported by a comprehensive non-clinical ocular toxicology program designed to support the use of FabA for IVT administration in clinical trials. Initial single-dose studies with FabA were conducted in rats and cynomolgus monkeys, with no ocular toxicity observed in either of these species. Based on similar findings in rats and monkeys, and in vitro pharmacology data, which showed monkeys to be more critical than rats, and sequence homology data, cynomolgus monkeys were selected for repeat-dose ocular toxicity studies of FabA.

[0259] Repeated-dose ocular toxicity studies included ophthalmoscopy (OE), IOP, electroretinogram (ERG), ocular histopathology, and measurement of FabA in serum and vitreous for TK analysis. Additionally, the PD properties of FabA were determined by measurement of C1q in vitreous (all repeated-dose studies), ocular tissues (two-dose studies), and inhibition of C1q-dependent hemolysis in serum (two-dose study).

[0260] Single-dose toxicity IVT administration of FabA was well tolerated in single-dose (rat and cynomolgus monkey) ocular toxicity studies, in which the NOAELs in rats and cynomolgus monkeys were considered to be 0.05 mg / eye (equivalent to a 10 mg human dose) and 5 mg / eye (equivalent to a 10 mg human dose), respectively, which were the maximum doses evaluated in each study and equivalent (2.5 mg / mL) when adjusted for vitreous volume (0.02 mL in rats and 2 mL in monkeys).

[0261] GLP single-dose ocular toxicity study of FabA via intravitreal injection in Sprague-Dawley rats In this single-dose GLP rat ocular toxicity study, young adult male rats were administered a single bilateral dose of vehicle or FabA by IVT injection at doses of 0.01 mg / eye (equivalent to a 2 mg human dose) and 0.05 mg / eye (equivalent to a 10 mg human dose). FabA-treated animals were sacrificed on days 1 (6 hours after dosing), 3, 7, 10, 20, and 30, and all vehicle control animals were sacrificed on day 30. All animals survived to scheduled necropsy.

[0262] The study included standard safety parameters. Blood samples were collected at sacrifice and vitreous samples were obtained at sacrifice for TK analysis. Additionally, ophthalmic examination (OE) including IOP, and ocular histopathology were evaluated.

[0263] No FabA-related changes were observed in any safety parameters evaluated, including OE, IOP, and ocular histopathology.

[0264] Vitreous exposure to FabA was determined by TK in animals treated with both 0.01 mg / eye (corresponding to a 2 mg human dose) and 0.05 mg / eye (corresponding to a 10 mg human dose) from 6 hours (first harvest) to 144 hours post-dose. Serum exposure to FabA was determined by TK in animals treated with 0.01 and 0.05 mg / eye (2-48 hours post-dose only).

[0265] No adverse events considered to be related to FabA were observed at any dose level observed in this study, including 0.05 mg / eye, the highest dose evaluated. Based on these results, the NOAEL was 0.05 mg / eye (2.5 mg / mL intravitreous).

[0266] A non-GLP single-dose ocular toxicity study of FabA via intravitreal injection in cynomolgus monkeys In this single-dose non-GLP cynomolgus monkey ocular toxicity study, young adult female cynomolgus monkeys were administered vehicle or FabA bilaterally via IVT injection at doses of 1 mg / eye (equivalent to a 2 mg human dose) and 5 mg / eye (equivalent to a 10 mg human dose). FabA-treated animals were sacrificed on days 1 (6 hours after dosing), 3, 7, 10, 20, and 30. All vehicle control animals were sacrificed on day 30, and all animals survived to scheduled necropsy.

[0267] The study evaluated standard safety parameters including OE, IOP and ocular histopathology. Additionally, blood samples were collected throughout the study and vitreous samples were analyzed at sacrifice for TK and PD.

[0268] FabA-related changes were limited to non-inflammatory, non-adverse findings, including histiocytic infiltration in the uvea and mild basophilia in the 1 mg / eye group. Findings at the 5 mg / eye dose consisted of histiocytic infiltration in the uvea and minimal to mild basophilia.

[0269] No FabA-related changes were observed in OE or IOP. No adverse events considered to be related to FabA were observed at any dose level in this study, including 5 mg / eye (the highest dose evaluated). Based on these results, the NOAEL was considered to be 5 mg / eye (2.5 mg / mL intravitreal).

[0270] Exposure to FabA in the vitreous was confirmed by TK throughout the study (through day 30) in all treated animals. Serum exposure to FabA was nonexistent at 1 mg / eye, low and transient at 5 mg / eye, and did not exceed 6 ng / mL (LLOQ 1.25 ng / mL). Clq was absent in the vitreous of all FabA-treated animals through day 30.

[0271] Repeated dose toxicity study In the repeat-dose ocular toxicity study, FabA was administered by IVT injection at least once every 4 weeks. Repeated administration of FabA in cynomolgus monkeys was well tolerated. In the first repeat-dose GLP ocular toxicity study, the NOAEL in cynomolgus monkeys was 5 mg / eye (equivalent to a human dose of 10 mg) at the highest dose evaluated, twice monthly. In a 26-week chronic ocular toxicity study in cynomolgus monkeys, adverse ocular changes were determined to be associated with the two injection procedures and / or ADA-mediated and not a direct effect of IVT administration of FabA. Thus, the NOAEL was determined to be 2.5 mg / eye (equivalent to a human dose of 5 mg) at 13 or 7 doses administered every other week or once monthly, respectively, in cynomolgus monkeys.

[0272] A 6-week GLP repeated-dose ocular toxicity study of FabA via intravitreal injection in cynomolgus monkeys The study included standard safety parameters and blood samples were collected throughout the study. Vitreous samples were collected at sacrifice for TK and PD analysis, as well as optic nerve sections at sacrifice for TK and PD analysis. Additionally, OE, IOP, ERG and ocular histopathology were evaluated.

[0273] Findings of FabA determined to be non-adverse were limited to a single high dose (2.5 mg / eye) (equivalent to a human dose of 5 mg) female who had minimal basophilic / blue staining of the vitreous without inflammation (termed basophilia). Importantly, no FabA-related changes were noted in OE, IOP, and ERG. Exposure to FabA in the vitreous of all treated animals was confirmed by TK during testing and recovery (day 30 after the last dose).

[0274] Serum exposure was not measurable at 1 mg / eye (corresponding to a 2 mg human dose) and was low and transient (12-48 hours after the first dose and 6-168 hours after the last dose) at 2.5 mg / eye (corresponding to a 5 mg human dose) and did not exceed 8 ng / mL (LLOQ 1.25 ng / mL). Absence of C1q in the vitreous of all treated animals was confirmed by PD when FabA concentrations were approximately 100 ng / mL. ADAs of FabA were detected in animals at doses of 1 mg / eye (corresponding to a 2 mg human dose) (6 of 12 animals) and 2.5 mg / eye (corresponding to a 5 mg human dose) (7 of 12 animals), but there was no clear effect of ADAs on serum or vitreous FabA exposure.

[0275] A 6-week GLP repeated-dose ocular toxicity study of FabA via intravitreal injection in cynomolgus monkeys In this 6-week GLP cynomolgus monkey ocular toxicity study, young adult male and female cynomolgus monkeys were administered vehicle or FabA bilaterally by IVT injection every 4 weeks (days 1 and 29) at a dose of 5 mg / eye (equivalent to a 10 mg human dose), followed by a 4-week recovery period. All main test animals were sacrificed on day 44 and all recovery animals were sacrificed on days 59 / 60. All main test and recovery animals survived to scheduled necropsy.

[0276] The study included standard safety parameters (except for systemic histopathology), and blood samples were collected throughout the study, as well as vitreous samples at sacrifice for TK and PD analysis. ADA and aqueous humor samples were collected and stored. Additionally, OE, IOP, ERG, and ocular histopathology were evaluated.

[0277] No FabA-related changes were observed in any safety parameters evaluated, including OE, IOP, ERG, and ocular histopathology. Minimal to mild basophilic / blue staining of the vitreous without inflammation (referred to as basophilia) was observed in both treated and control animals and was therefore considered not associated with FabA.

[0278] Exposure to FabA in the vitreous of all treated animals was confirmed by TK during the study and recovery (30 days after the last dose). Absence of FabA was confirmed in the serum and vitreous of control animals. Absence of C1q in the vitreous was confirmed by PD on day 44 in all treated main study animals. On day 59, two of four recovery animals had measurable C1q in the vitreous. ADA of FabA was detected in animals (9 of 10 animals) in the 5 mg / eye (corresponding to a 10 mg human dose) dose group, but there was no clear effect of ADA on serum or vitreous FabA exposure.

[0279] >80% inhibition of C1q-dependent hemolysis was achieved 24–48 h after FabA administration, after which the effect returned to baseline.

[0280] At day 44, C1q concentrations in the retina, choroid and optic nerve head were also significantly reduced and at day 59 continued to decrease in the retina and choroid, but not in the optic nerve head.

[0281] In this study, no adverse events potentially related to FabA were observed at any dose level, including 5 mg / eye (equivalent to a 10 mg human dose), the highest dose evaluated. Based on these results, the NOAEL was 5 mg / eye (equivalent to a 10 mg human dose) (2.5 mg / mL intravitreal).

[0282] A 26-week GLP repeated-dose ocular toxicity study of FabA via intravitreal injection in cynomolgus monkeys with a 10-week recovery period In this 26-week GLP cynomolgus monkey ocular toxicity study, young adult male and female cynomolgus monkeys received vehicle or FabA bilaterally via IVT injection at doses of 2.5 mg / eye once monthly (equivalent to a 5 mg human dose), 5 mg / eye once monthly (equivalent to a 10 mg human dose), and 5 mg / eye once every other week, followed by a 10-week recovery period. A single injection of 50 μL equivalent to 2.5 mg / eye, or two injections totaling 100 μL (two 50 μL injections 10 minutes apart equivalent to 5 mg / eye) were administered every 2 weeks (13 dosing periods) or every 4 weeks (7 dosing periods). All main study animals were sacrificed on day 184 and all recovery animals were sacrificed on days 242 / 243. All main study and recovery animals survived to scheduled necropsy.

[0283] Animals in the control group, the monthly 5 mg / eye (equivalent to a 10 mg human dose), and the biweekly 5 mg / eye groups experienced dosing days or dosing cessations. The double injections in these groups were halted due to adverse findings detected by OE, which were deemed related to the procedure and high dose volume. Beginning on day 71 of the study, the monthly 5 mg / eye group received 2.5 mg / eye (equivalent to a 5 mg human dose) once a month (referred to as monthly 5 / 2.5 mg / eye), and the biweekly 5 mg / eye group received 2.5 mg / eye every other week (referred to as biweekly 5 / 2.5 mg / eye). Dosing days continued after the double injections were stopped in these groups (including the control group). The cessation of double injections was related to the procedure and / or ADA, as described below. There were no dosing days in the monthly 2.5 mg / eye group (low dose group).

[0284] The study included standard safety parameters (except for systemic histopathology), and blood samples were collected throughout the study, as well as vitreous samples at sacrifice for TK and PD analysis. ADA and aqueous humor samples were collected and stored. Additionally, OE, IOP, ERG, ocular histopathology, and immunohistochemistry (IHC) for detection of intraocular deposited immune complexes were evaluated.

[0285] No FabA-related changes were observed in body weight, food consumption, electroretinography, intraocular pressure measurements, or clinical pathology.

[0286] Ocular clinical signs and ocular examination findings considered to be related to FabA were limited to ocular opacification (presumably due to opacification in the anterior chamber, lens capsule, and / or posterior chamber) and the presence of cells and / or pigment. The presence of these findings in animals with no detectable ADA in the serum (4 of 12 animals in group 2, 2 of 12 animals in group 3, and 2 of 12 animals in group 4) indicates an association with FabA. Findings considered to be related to ADA and potentially immune complex deposition tended to be more severe and included aqueous humor flare and the presence of vitreous opacification, changes in the pupillary light reflex, and retinal vascular attenuation.

[0287] After IVT administration of FabA to male and female monkeys, systemic exposure, as measured by serum FabA concentrations, was transient and low, not exceeding 86.3 ng / mL after dosing on day 85, or 60.81 ng / mL after the last dose on day 169. Exposure to FabA was confirmed by TK in almost all treated animals receiving FabA through day 169, which was consistent with undetectable vitreous C1q, except in some animals on dose-free days. On day 242 / 243, after a 10-week treatment-free recovery period, FabA vitreous concentrations were not measurable, and C1q concentrations were detectable in all treatment dose groups. Absence of FabA was confirmed in the serum and vitreous of control animals.

[0288] The presence of anti-FabA antibodies in serum samples was confirmed in 4 of 12 Group 1 (control) animals, 8 of 12 Group 2 animals, 10 of 12 Group 3 animals, and 10 of 12 Group 4 animals. Two Group 1 animals were confirmed as positive at one time point per animal after day 1, while FabA-treated animals were identified as ADA positive at three or more time points (a total of four or five samples were collected for main study and recovery animals, respectively). There was no clear effect of ADA on serum or intravitreal FabA exposure.

[0289] At terminal euthanasia on day 184, microscopic changes consistent with an ADA-mediated immune response to FabA were observed in right eyes of the monthly and biweekly 5 / 2.5 mg / eye groups (medium and high dose groups, respectively). Inflammation-related intraocular changes included mild mixed cell infiltration in the ciliary body and vitreous cavity, minimal to moderate fibrosis in the vitreous cavity (severity proportional to dosing frequency), and minimal to mild posterior lens degeneration (severity proportional to dosing frequency). Minimal perivascular mononuclear cell infiltration was also observed within the posterior retina in one female each of the biweekly and monthly 5 / 2.5 mg / eye treatment groups. Minimal to moderate mononuclear cell infiltration was also observed within the peribulbar limbus of animals treated with 5 / 2.5 mg / eye biweekly and monthly, with severity proportional to dosing frequency.

[0290] At euthanasia after recovery on day 242 / 243, microscopic changes in the right eye related to an ADA-mediated immune response to FabA were limited and mild in the monthly 5 / 2.5 mg / eye group, whereas further changes persisted or occurred in the biweekly 5 / 2.5 mg / eye group. Minimal histiocyte infiltration of the vitreous cavity and uvea, as well as increased basophilia in the vitreous cavity, were limited during the recovery phase in animals receiving biweekly and / or monthly FabA 5 / 2.5 mg / eye. These changes were similar to those observed in control animals at terminal necropsy and were considered to be related to mild inflammation / destruction of the anterior vitreous secondary to the IVT injection procedure. However, the persistence of these changes after the recovery period, their resolution in recovery control animals, and the presence of more severe inflammatory changes at terminal necropsy in animals receiving FabA at 5 / 2.5 mg / eye every two weeks and every month, indicated that these changes during recovery may represent resolution of inflammation associated with an ADA-mediated immune response to FabA, rather than residual effects of the injection procedure.Minimal mononuclear cell infiltration in the peribulbar limbus persisted in both groups receiving 5 / 2.5 mg / eye every two weeks and every month.

[0291] In the biweekly 5 / 2.5 mg / eye group, minimal mixed cell infiltration and fibrosis in the vitreous cavity persisted, while mild loss of retinal cellularity and hemosiderin pigmentation occurred. Minimal perivascular mononuclear cell infiltration in the retina and optic disc was also observed in the biweekly 5 / 2.5 mg / eye group. These changes were considered secondary to an ADA-mediated response to FabA.

[0292] Adverse pathological microscopic changes were deemed secondary to ADA-mediated inflammation and included intravitreal fibrosis, lens degeneration, and reduced retinal cellularity in the biweekly and monthly 5 / 2.5 mg / eye groups.

[0293] In the 2.5 mg / eye monthly group, no FabA-related microscopic changes were noted either at terminal or post-recovery euthanasia.

[0294] Immunohistochemistry was performed on 2 of 12, 4 of 12, and 6 of 12 animals from Groups 1, 3, and 4, respectively. Evaluation revealed the presence of granular deposits containing FabA, monkey IgG, IgM, and / or C3 detected immunohistochemically in the left eyes of 4 of 10 treated animals in the medium dose, i.e., monthly 5 / 2.5 mg / eye group (2 of 4 animals), and the high dose, i.e., biweekly 5 / 2.5 mg / eye group (2 of 6 animals) selected for IHC. These intravascular deposits were present in association with a perivascular inflammatory cell infiltrate, similar to the deposits observed by hematoxylin-eosin evaluation of the right eye. Other microscopic changes observed in the right eye were consistent with secondary changes associated with an immune response to this FabA in the monkeys. Ocular immune complex deposits were not observed in all animals selected for immunohistochemistry, including some of the serum ADA-negative animals, which was not unexpected since identification of deposits can vary with tissue sectioning and serum ADA is not always present in animals with microscopic evidence consistent with immune complex pathology. Additionally, some animals with multiple dosing-free days may have cleared ADA and / or immune complexes prior to analysis. The presence of immunohistochemically confirmed deposits was considered the most compelling weight of evidence that the similar pathogenetically consistent pathology observed in the right eye, even in a subset of animals, is likely related to a FabA immune response.

[0295] In this 26-week chronic ocular toxicity study in cynomolgus monkeys, adverse ocular changes were determined to be associated with the two injection procedures and / or ADA-mediated, and not a direct effect of IVT administration of FabA. Therefore, the NOAEL was determined to be 2.5 mg / eye (equivalent to a 5 mg human dose) for 13 or 7 doses administered biweekly or monthly, respectively, in cynomolgus monkeys.

[0296] Example 9: Evaluation of FabA in clinical trials FabA drug product is a sterile isotonic solution for IVT injection. A Phase 1: first-in-human, open-label, dose-escalation study (FabA-GLA-01) was conducted to evaluate the initial safety and tolerability of a single IVT injection of FabA in patients with primary open-angle glaucoma.

[0297] A Phase 1b: randomized, double-blind study (FabA-GLA-02) was conducted to evaluate the safety and tolerability of repeated IVT injections of FabA in patients with primary open-angle glaucoma.

[0298] Results from both studies showed that single (1-5 mg / eye) (corresponding to a human dose of 2-10 mg) and repeated (2.5 and 5 mg / eye, administered twice 4 weeks apart) doses of FabA via IVT were found to be well tolerated in glaucoma patients, with no serious or significant adverse events (AEs) reported. Ocular AEs in patients treated with FabA in these studies included conjunctival hyperemia, conjunctival hemorrhage, and ocular irritation, which occurred only in the treated eye. In the Phase 1b study, ocular AEs in patients in the sham group included eye pain, ocular foreign body sensation, ocular hyperemia, and blurred vision. No systemic AEs considered related to the FabA IVT treatment occurred.

[0299] A single IVT injection of 2.5 mg (corresponding to a 5 mg human dose) and 5 mg (corresponding to a 10 mg human dose) of FabA inhibited free C1q in aqueous humor for at least 29 days (Study FabA-GLA-02).

[0300] Pharmacokinetics and Pharmacodynamics in Humans Ocular Pharmacokinetics and Pharmacodynamics FabA-GLA-02 is a Phase 1b study in which aqueous humor was collected to evaluate PK and PD. Subjects received two IVT injections of sham, 2.5 mg / eye FabA (corresponding to a 5 mg human dose), or 5 mg / eye FabA (corresponding to a 10 mg human dose), 29 days apart. In this study, aqueous humor was collected pre-dose and 29 days after the first FabA dose and before the second dose. On day 29 (D29), free FabA was detected in the aqueous humor of all treated patients. In parallel, both dose levels of 2.5 mg / eye and 5 mg / eye FabA inhibited free C1q in the fluid for at least 29 days (Figure 12).

[0301] Systemic Pharmacokinetics and Pharmacodynamics FabA-GLA-01 is a single-dose Phase 1 study in which serum FabA and C1q were collected pre-dose and 3 hours post-dose. FabA-GLA-02 is a multiple-dose Phase 1b study in which serum and FabA and C1q were collected pre-dose and 3 hours post-dose after each of two doses, 29 days apart. FabA was not generally detectable in the systemic circulation after single or repeated IVT injections at any dose level tested in the Phase 1 or Phase 1b clinical trials. Similarly, no changes in circulating free C1q were detected in either study.

[0302] As described below, a dose level of 5 mg / eye (corresponding to a 10 mg human dose) was well tolerated when administered as a single dose or two doses 29 days apart in FabA clinical trials. As described above, in a Phase 1b study, single doses of 2.5 mg (corresponding to a 5 mg human dose) and 5 mg (corresponding to a 10 mg human dose) of FabA inhibited free C1q in body fluids for at least 29 days (Figure 12).

[0303] Safety and Effectiveness Phase 1 dose escalation study (FabA-GLA-01) This was a Phase 1, open-label, dose-escalation study evaluating the safety / tolerability and PK of a single IVT injection of FabA in patients with primary open-angle glaucoma. Eligible patients were adults who had reliable visual field testing in the study eye using the Humphrey Field Analyzer-Swedish Interactive Threshold Algorithm (HFA-SITA) 24-2 fast algorithm with a cutoff of 33% for fixation failure and 33% for false-positive response rate, had a mean deviation of 3-18 dB on reliable visual field testing, and had an IOP of <21 mmHg in the study eye on a stable IOP treatment regimen for ≥4 weeks prior to dosing. Nine patients were assigned to three cohorts, with three patients per cohort enrolled as follows: Cohort 1 = 1.0 mg / eye, single dose (0.02 mL) x 1 dose Cohort 2 = 2.5 mg / eye, single dose (0.05 mL) x 1 dose Cohort 3 = 5.0 mg / eye, single dose (0.10 mL) x 1 dose

[0304] After screening, three eligible patients were enrolled in the lowest open cohort and enrolled in the next cohort, which began only after tolerability and short-term safety were demonstrated at the aforementioned lower dose. All patients in each cohort were required to complete a minimum 15-day safety observation period before being eligible to receive injections in the next cohort. No dose-limiting toxicities (DLTs) were reported during the study.

[0305] Nine patients were enrolled, treated, and completed the study.

[0306] safety Ocular treatment-emergent adverse events (TEAEs) included conjunctival hyperemia (all dose levels), conjunctival hemorrhage (2.5 mg / eye only), and ocular irritation (1 mg / eye only) and occurred only in the study eye.

[0307] The only systemic TEAE experienced in this study was sinusitis. All TEAEs were mild in severity. No serious or significant TEAEs were observed. No patients discontinued treatment or withdrew from the study due to TEAEs. • In 9 of 9 patients, IOP returned to normal (within 5mmHg of IOP just before injection or <21mmHg) within 30 minutes. • No patients showed any evidence of anti-FabA antibodies.

[0308] Overall summary / conclusion: In this study, single IVT administration of FabA was well tolerated up to 5 mg / eye in patients with stable glaucoma. Ocular AEs reported were similar to those reported with IVT administration of approved agents. No safety signals were observed for FabA.

[0309] FabA was not normally detectable in the systemic circulation, and no changes in circulating free C1q were detected after a single IVT administration.

[0310] Phase 1b (FabA-GLA-02) This was a double-blind, randomized, sham-controlled study to evaluate two dose levels of FabA administered as repeated IVT injections versus sham injections in patients with primary open-angle glaucoma. Eligible patients were adults who had reliable visual field testing in the study eye using the HFA-SITA fast algorithm with a cutoff of 33% for fixation failure and 33% for false-positive response rate, a mean deviation of -3 to -24 dB in the study eye, an IOP of <21 mmHg at screening and on Day 1, who were on a stable IOP treatment regimen for ≥4 weeks prior to injection, and who had no anticipated changes in IOP treatment regimen during the study. Patients received two injections, 4 weeks apart, and were followed for a total of 12 weeks for evaluation of safety, tolerability, PK, PD, immunogenicity, and ongoing exploratory evaluations. Patients were randomly assigned (1:1:1) to one of three cohorts (designated 5 patients per cohort) as follows: Dose level 1 = 2.5 mg / eye, single dose (0.05 mL) x 2 doses Dose level 2 = 5.0 mg / eye, single dose (0.10 mL) x 2 doses Sham = 0 mg / eye x 2 doses

[0311] Eighteen patients were randomized (7 to the 2.5 mg FabA group, 5 to the 5.0 mg FabA group, and 6 to the sham group) and 17 patients received treatment. One patient in the 2.5 mg group was randomized but did not receive treatment. Sixteen patients completed the study.

[0312] safety Ocular TEAEs experienced by patients treated with FabA included conjunctival hyperemia (2.5 and 5 mg / eye), conjunctival hemorrhage (5 mg / eye only), and eye irritation (5 mg / eye only). None of these TEAEs were experienced by patients in the sham group. Ocular TEAEs in patients in the sham group included eye pain, foreign body sensation in the eye, eye hyperemia, and blurred vision, each occurring in one patient.

[0313] Systemic TEAEs were observed during the study, but none were considered by the investigators to be related to study treatment. All TEAEs were mild in severity. All but one of the reported TEAEs occurred after the first dose and before the second administration of study treatment. No serious or significant TEAEs were observed. No patients discontinued treatment or withdrew from the study due to TEAEs. IOP returned to normal (<21 mmHg) within 30 minutes of IVT injection in 16 of 17 patients and within 45 minutes in the remaining patient. Of the 11 patients who received intravitreal FabA, 6 patients tested were positive for at least one time point. One patient was ADA positive with a modest increase in titer over time, and the remaining 5 patients were ADA positive at all time points, including before administration, and titers did not change over time. One sham patient was ADA positive at all time points, including before administration, and titers did not change over time. Collectively, these data suggest that the relationship between ADA measurements and FabA administration is unclear.

[0314] Overall summary / conclusion: In patients with stable glaucoma, two IVT doses of FabA administered 4 weeks apart were well tolerated up to 5 mg / eye. Ocular AEs reported were similar to those reported with IVT administration of approved agents. No safety signals for FabA were observed in this study.

[0315] A single IVT dose of FabA (2.5 and 5 mg / eye) inhibited free C1q in aqueous humor for at least 29 days.

[0316] Example 10: A Phase 2, Multicenter, Randomized, Parallel-Group, Double-Masked, 4-Arm, Sham-Controlled Study of the Efficacy, Safety, and Tolerability of FabA Administered by Intravitreal Injection in Patients with Geographic Atrophy (GA) Secondary to Age-Related Macular Degeneration (AMD) basis overview The study will be conducted in patients with GA secondary to AMD. The purpose of the study is to determine whether intravitreal (IVT) injections of FabA once a month (EM) or once every other month (EOM) for 12 months will reduce the growth rate of GA lesions. The study will consist of a 30-day screening period and a 12-month treatment period followed by a 6-month (treatment-free) follow-up period. Patients will participate for a total of 19 months. Patients will return to the clinic monthly for treatment and / or safety assessments during the 12-month treatment period.

[0317] Approximately 240 patients were enrolled and randomly assigned to one of four treatment arms, with approximately 204 patients evaluable for the primary analysis at Month 12 (primary analysis was based on modified intention-to-treat [ITT]).

[0318] Intervention group and duration: Allocation of study interventions was on a randomized basis (2:2:1:1). Patients were assigned to one of the following treatment arms: Dose levels were fixed and would not change. Arm 1 = FabA 5.0 mg / eye (0.10 mL) once monthly (EM) for 12 months (12 doses) Arm 2 = FabA 5.0 mg / eye (0.10 mL) once every other month (EOM) for 12 months (6 doses) Arm 3 = sham injections with EM for 12 months (12 sham injections) Arm 4 = Sham injections with EOM for 12 months (6 sham injections)

[0319] injection FabA / sham administration is completed using aseptic technique by the physician administering the injection.

[0320] All patients randomized to FabA will receive 5.0 mg / eye IVT (fixed volume of 0.10 mL) once monthly or once every other month for 12 months.

[0321] After injection Immediately after drug administration, the injecting physician evaluates whether manual valve vision or central retinal artery perfusion can be seen. If necessary, rule out other causes of visual impairment such as vitreous hemorrhage. If necessary, perform digital massage and administer topical / oral IOP-lowering medication until manual valve vision or central retinal artery perfusion is observed.

[0322] IOP (intraocular pressure measurement) is assessed only in the study eye 30 minutes after drug administration and, if elevated, every 15 minutes thereafter until IOP<25 mmHg.

[0323] Pharmacokinetics, Pharmacodynamics and Immunogenicity Blood samples for PK (FabA serum concentrations) and PD (serum C1q and plasma concentrations of other biomarkers) assessments will be collected at patient visits, 30 minutes pre-dose and within 3 hours (±15 minutes) post-dose.

[0324] During the patient's clinic visit, samples for immunogenicity testing (ADA) will be collected prior to injection. Additionally, samples for ADA will be collected at week 2 during a clinic or home visit.

[0325] Pharmacokinetics: Serum is required for this study. Blood samples will be collected to measure serum concentrations of FabA.

[0326] Pharmacodynamics: Serum and plasma are required for this study. Serum concentrations of C1q and plasma concentrations of exploratory complement biomarkers will be analyzed.

[0327] Immunogenicity: Serum is required for this study. Immunogenicity will be assessed by analysis of serum anti-drug (FabA) antibodies (ADA).

[0328] Intravitreal injection procedure Preparation of FabA From a sterile vial of FabA, the entire volume of FabA (approximately 0.3 mL) is withdrawn using a sterile 1.0 cc syringe fitted with a 19 gauge x 1-1 / 2 inch, 5 micron filter needle.

[0329] The filter needle is replaced with a 30 gauge×½ inch injection needle. Immediately prior to injection, the excess volume of FabA is expelled from the syringe leaving only the required injection volume in the syringe. The dose volume of FabA will be fixed at 0.10 mL, once every month (EM) for 12 months (12 doses) or once every other month (EOM) for 12 months (6 doses).

[0330] Preparation for intravitreal injection 1. Examine the test eye. 2. Measure and record preoperative intraocular pressure (IOP) in the study eye prior to injection. Tonometry will be performed in the study eye only. IOP must be ≦21mmHg to proceed. If >21mmHg, FabA injection will be rescheduled and IOP managed at the investigator's discretion. 3. If necessary, apply one drop of topical phenylephrine chloride eye drops 2.5% to the test eye 30 minutes prior to injection to allow visualization of the posterior pole after injection. 4. Just before injection: • Have the patient lie flat on the examination chair with the neck well supported.

[0331] Intravitreal injection 1. Hand washing, sterile gloves and a surgical mask are required for injections. 2. Apply topical proparacaine 0.5% to the test eye. 3. Apply povidone iodine 10% to the eyelashes and lid margin. Avoid extensive massaging of the eyelids either before or after injection to avoid exposing the meibomian glands. 4. Hold the eyelid away from the intended injection site during the procedure. Use of a microscope is recommended. 5. Apply povidone-iodine 5% to the conjunctival surface, including the intended injection site. 6. Injection of FabA: Insert the needle perpendicular to the sclera, 3.5-4 mm posterior to the limbus, between the vertical and horizontal rectus muscles. Immediately after withdrawing the needle, apply a sterile cotton swab applicator over the injection site to reduce vitreous reflux. 7. Sham Injection: Preparation and post-injection care for sham injections are the same as for injections with Fab A. Sham injections are performed in the typical vitreous injection location using the blunt needle of an empty syringe without a needle to apply pressure to the eye.

[0332] After intravitreal injection 1. Patients will remain in the clinic following injection for ocular evaluation and safety follow-up. 2. Immediately assess manual valve vision or central retinal artery perfusion and rule out other causes of vision loss such as vitreous hemorrhage. If no other causes are found, perform digital massage and administer topical / oral IOP-lowering medications until manual valve vision or central retinal artery perfusion is observed. 3. In the study eye, IOP measurements are obtained only 30 minutes after injection and, if elevated, every 15 minutes until IOP<25mmHg. Intraocular pressures above 30mmHg for more than 15 minutes should be treated at the physician's discretion. 4. Topical antibiotics are not necessary.

[0333] Example 11: C1q mediates microglial pruning of photoreceptor synapses in a light damage model of photoreceptor degeneration Anti-C1q treatment (e.g., Mab3) reduces retinal complement levels (Figures 5A-5D), reduces inflammation (Figures 13A and 13B), and reduces neurodegeneration (Figures 13A and 13C-13D) in a light injury model. Figures 13A-13D show immunofluorescence (IF) data. Figure 13B shows reduced microgliosis in the outer plexiform layer (OPL) (also known as the outer synaptic layer) of the retina 3 days after treatment. Reduced microgliosis is associated with reduced inflammation. Figure 13C shows significant preservation of photoreceptor synapses and Figure 13D shows significant preservation of cell bodies 5 days after treatment. Post-treatment measurements of preserved photoreceptor synapses and cell bodies demonstrate that treatment with anti-C1q treatment reduces neurodegeneration in this light injury model.

[0334] Example 12: Intravitreal anti-C1q treatment reduced retinal complement component levels in the rd10 mouse model. Measurable therapeutic (e.g., Mab3) levels were found in retinal lysates from animals that received anti-C1q treatment, but not in IgG1-treated or untreated groups (Figure 14A). Increased levels of classical complement component C1q were confirmed in retinal lysates from both untreated and IgG d10 animals compared to WT (Figure 14B). Anti-C1q treatment resulted in reduced C1q levels in retinal lysates compared to IgG1-treated and untreated rd10 groups (Figure 14B), confirming good measurable PK and C1q involvement in the retina (and in plasma, data not shown). Figure 15A is a bar graph depicting quantification of immunofluorescence images, and Figure 15B shows immunofluorescence images showing preservation of photoreceptor synapses (BSN markers) upon treatment with C1q inhibitors. This is evidence of photoreceptor synapse protection.

[0335] Incorporation by Reference Each of the patents, published patent applications, and non-patent publications cited herein is hereby incorporated by reference in its entirety.

[0336] 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. A composition comprising an anti-C1q antibody or its antigen-binding fragment for the treatment of hereditary retinal disease in human patients, for intravitreal injection at doses of about 1 mg to about 10 mg of the anti-C1q antibody or its antigen-binding fragment, The composition wherein the anti-C1q antibody or its antigen-binding fragment comprises a light chain variable domain having 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, and a heavy chain variable domain having 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.

2. The composition according to claim 1, wherein the hereditary retinal disease is retinitis pigmentosa, colloideremia, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis, X-linked RP, or Usher syndrome.

3. A composition comprising an anti-C1q antibody or its antigen-binding fragment for the treatment of retinal detachment in human patients, for intravitreal injection at doses of about 1 mg to about 10 mg of the anti-C1q antibody or its antigen-binding fragment, The composition wherein the anti-C1q antibody or its antigen-binding fragment comprises a light chain variable domain having 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, and a heavy chain variable domain having 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 composition according to claim 3, wherein the anti-C1q antibody or its antigen-binding fragment is to be administered before, after, or concurrently with retinal detachment surgery.

5. The composition according to any one of claims 1 to 4, which restores or improves the visual acuity of the human patient.

6. The composition according to any one of claims 1 to 4, wherein the anti-C1q antibody or its antigen-binding fragment comprises a light chain variable domain having an amino acid sequence having at least about 95% homology with an amino acid sequence selected from SEQ ID NOs. 4 and 35 to 38.

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

8. The composition according to any one of claims 1 to 4, wherein the anti-C1q antibody or its antigen-binding fragment comprises a heavy chain variable domain having an amino acid sequence having at least about 95% homology with an amino acid sequence selected from SEQ ID NOs. 8 and 31 to 34.

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

10. The composition according to any one of claims 1 to 4, wherein the anti-C1q antibody or its antigen-binding fragment is a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, or an antibody derivative thereof.

11. The composition according to any one of claims 1 to 4, wherein the antigen-binding fragment is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, a diabody, or a single-chain antibody molecule.

12. The composition according to claim 11, wherein the Fab fragment comprises the heavy chain Fab fragment of SEQ ID NO: 39 and the light chain Fab fragment of SEQ ID NO:

40.

13. The composition according to any one of claims 1 to 4, which is to be administered once a week, once every two weeks, once every three weeks, once a month, once every four weeks, once every six weeks, once every eight weeks, once every two months, once every ten weeks, once every twelve weeks, once every three months, or once every four months.

14. The composition according to claim 13, which is to be administered for at least three months, at least four months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months.

15. The composition according to claim 13, which is intended to be administered for 12 months.

16. The composition according to any one of claims 1 to 4, comprising approximately 1 mg, approximately 1.5 mg, approximately 2 mg, approximately 2.5 mg, approximately 3 mg, approximately 3.5 mg, approximately 4 mg, approximately 4.5 mg, approximately 5 mg, approximately 5.5 mg, approximately 6 mg, approximately 6.5 mg, approximately 7 mg, approximately 7.5 mg, approximately 8 mg, approximately 8.5 mg, approximately 9 mg, approximately 9.5 mg, or approximately 10 mg of the anti-C1q antibody or its antigen-binding fragment.

17. The composition according to any one of claims 1 to 4, wherein the aforementioned dose comprises about 1 mg of the anti-C1q antibody or its antigen-binding fragment.

18. The composition according to any one of claims 1 to 4, wherein the aforementioned dose comprises about 2.5 mg of the anti-C1q antibody or its antigen-binding fragment.

19. The composition according to any one of claims 1 to 4, wherein the aforementioned dose comprises about 5 mg of the anti-C1q antibody or its antigen-binding fragment.

20. The composition according to any one of claims 1 to 4, wherein the aforementioned dose comprises about 2 mg of the anti-C1q antibody or its antigen-binding fragment.

21. The composition according to any one of claims 1 to 4, wherein the aforementioned dose comprises approximately 7.5 mg of the anti-C1q antibody or its antigen-binding fragment.

22. The composition according to any one of claims 1 to 4, wherein the aforementioned dose comprises about 10 mg of the anti-C1q antibody or its antigen-binding fragment.

23. The composition according to any one of claims 1 to 4, wherein the aforementioned dose comprises the anti-C1q antibody or its antigen-binding fragment in an amount of about 1 mg to about 2.5 mg, about 2.5 mg to about 5 mg, about 5 mg to about 7.5 mg, or about 7.5 mg to about 10 mg.

24. Use of an anti-C1q antibody or its antigen-binding fragment for the manufacture of a pharmaceutical product for the treatment of hereditary retinal disease in human patients, wherein the pharmaceutical product is for intravitreal injection in a dose of about 1 mg to about 10 mg of the anti-C1q antibody or its antigen-binding fragment, The composition wherein the anti-C1q antibody or its antigen-binding fragment comprises a light chain variable domain having 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, and a heavy chain variable domain having 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.

25. Use of an anti-C1q antibody or its antigen-binding fragment for the manufacture of a pharmaceutical product for the treatment of retinal detachment in a human patient, wherein the pharmaceutical product is for intravitreal injection in a dose of about 1 mg to about 10 mg of the anti-C1q antibody or its antigen-binding fragment, The composition wherein the anti-C1q antibody or its antigen-binding fragment comprises a light chain variable domain having 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, and a heavy chain variable domain having 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.

Images