Compositions and methods for treating ocular diseases

Abstract

The present disclosure relates generally to compositions and methods for preventing, reducing the risk of developing or treating hereditary retinal disease (IRD) (e.g., retinitis pigmentosa, choroidal defect, fundus yellow spot, cone-rod dystrophy, Leeb congenital amaurosis), X-linked RP and Erhe syndrome or retinal detachment.

Description

[0001] Related Applications

[0002] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 336,539, filed on April 29, 2022, which is hereby incorporated by reference in its entirety. Background Art

[0003] Inherited retinal diseases are a group of conditions that can cause severe vision loss or even blindness. Each IRD is caused by at least one gene that is not functioning properly. IRDs (such as retinitis pigmentosa) can affect individuals of all ages, can develop at different rates, and are rare. However, most are progressive, meaning that the symptoms of the disease worsen over time. Current treatments aim to repair the genetic defect, but there are many genetic defects and each defect affects only a small number of patients. The only approved treatment is voretigene neparvovec-rzyl, which works for the gene mutation RPE65, but this gene only appears in 1-2% of IRD patients. Another approach is to use a device called a "retinal prosthesis" that converts light into electrical energy to directly stimulate the retina, but this approach does not address the pathophysiology of the disease. Currently, there are no approved treatments or therapies for IRD that are gene-agnostic. Therefore, there is a significant unmet need for treatments for patients with IRD.

[0004] Retinal detachment is an eye condition in which the retina peels away from the supporting tissue layer beneath it. Retinal detachment occurs in approximately 10-12 cases per 100,000 people each year. In approximately 50% of cases, central retinal detachment leads to macular detachment of the retina. When the central retina detaches, visual recovery only reaches approximately 50% of pre-detachment vision, despite successful reattachment of the retina. The reason for this limited visual recovery is the degeneration of the photoreceptor cells. There are no approved treatments or therapies to improve visual function after successful macular detachment retinal detachment surgery. Therefore, there is a significant unmet need for treatments for patients with retinal detachment. Summary of the invention

[0005] 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, choroidal coloboma, Stargardt disease, cone-rod dystrophy, Leber congenital amaurosis, X-linked RP, Usher Syndrome) and / or retinal detachment in human patients. In some embodiments, anti-C1q antibodies are administered before, after, and / or concurrently with retinal detachment surgery. These methods can restore vision in human patients and / or improve vision in human patients.

[0006] Such methods comprise administering to the patient by intravitreal injection a composition comprising about 1 mg to about 10 mg of an anti-C1q antibody, wherein the antibody comprises a light chain variable domain comprising HVR-L1 having an amino acid sequence of SEQ ID NO:5, HVR-L2 having amino acids of SEQ ID NO:6, and HVR-L3 having amino acids of SEQ ID NO:7, and a heavy chain variable domain comprising HVR-H1 having an amino acid sequence of SEQ ID NO:9, HVR-H2 having amino acids of SEQ ID NO:10, and HVR-H3 having amino acids of SEQ ID NO:11. In some embodiments, the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, and wherein the light chain variable domain comprises HVR-L1 having an amino acid sequence of SEQ ID NO: 5, HVR-L2 having amino acids of SEQ ID NO: 6, and HVR-L3 having amino acids of SEQ ID NO: 7. 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, and wherein the heavy chain variable domain comprises HVR-H1 having an amino acid sequence of SEQ ID NO: 9, HVR-H2 having amino acids of SEQ ID NO: 10, and HVR-H3 having amino acids of SEQ ID NO: 11. In some embodiments, the heavy chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 31-34. In some embodiments, the antibody comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 35-38, and wherein the light chain variable domain comprises HVR-L1 having an amino acid sequence of SEQ ID NO: 5, HVR-L2 having amino acids of SEQ ID NO: 6, and HVR-L3 having amino acids of SEQ ID NO: 7, the heavy chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 31-34, and wherein the heavy chain variable domain comprises HVR-H1 having an amino acid sequence of SEQ ID NO: 9, HVR-H2 having amino acids of SEQ ID NO: 10, and HVR-H3 having amino acids of SEQ ID NO: 11.In some embodiments, the antibody comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprises an amino acid sequence selected from SEQ ID NO: 4 and 35-38, and the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NO: 8 and 31-34. The antibody can be a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, an antibody fragment or an antibody derivative thereof. The antibody fragment can be a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a 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.

[0007] In some embodiments, the antibody is administered once a week, once every other week, once every three weeks, once a month, once every 4 weeks, once every 6 weeks, once every 8 weeks, once every other month, once every 10 weeks, once every 12 weeks, once every three months, or once every 4 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.

[0008] 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 the anti-C1q antibody. The composition administered may comprise about 1 mg to about 5 mg of the 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 the anti-C1q antibody. The composition administered may comprise about 5 mg of the anti-C1q antibody. The composition administered may comprise about 10 mg of the anti-C1q antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figures 1A-1F Photoreceptor cell damage and microgliosis following light exposure are shown. Figures 1A-1C Shown are immunofluorescence (IF) images and quantification showing progressive loss of photoreceptor cell synapses (Bassoon) and cell bodies (Dapi) following light damage. Figures 1D-1FIF images and quantification of increased microglia / macrophage reactivity (Iba1 and CD68) following photodamage are shown. The distribution of phagocytic microglia in the synaptic layer peaks at day 1, when significant synaptic loss is first observed.

[0010] Figure 2A-2E Complement signaling and C1q distribution in the retina after light exposure are shown. Figure 2A-2C An ELISA assay is shown that demonstrates increased levels of the initial classical complement components C1q and C1s and the downstream activation product C3d in retinal lysates following light damage. Figure 2D IFs showing retinal C1q distribution and its colocalization with microglia / macrophages (Iba1) and synapses (Bassoon) are shown. Figure 2E Correlation analysis is shown, which shows a significant negative correlation between C1q levels in the OPL and photoreceptor cell synaptic density, consistent with a causal relationship.

[0011] Figure 3 A-3E shows microglial synaptic phagocytosis after light exposure. Figure 3 A shows IFs showing increased levels of C1q in the OPL of light-damaged retinas and its proximity to bassoon +ve synapses (i). Figure 3 B shows a high-resolution and 3D surface-rendered image showing engulfment of C1q-labeled synapses by microglia in the photodamaged retina. Figure 3 C-3E shows a quantitative analysis showing that synaptic density was significantly reduced in light-damaged retinas compared to undamaged ones ( Figure 3 C) The percentage of C1q-labeled synapses increased ( Figure 3 D) Increased microglia engulfing C1q-labeled synapses ( Figure 3 E).

[0012] Figure 4 A-4F shows phosphatidylserine (PS) externalization at photoreceptor cell synapses and binding to C1q in vitro. Figure 4 A shows IF showing PSVue labeling of PS in the OPL of light-damaged retina. 3D surface rendering images (i-ii) show PSVue close to Bassoon and C1q, indicating PS externalization at the synapse. Figure 4 B shows an assay showing binding of CIq to PS lipid beads. No binding to control phosphatidylcholine (PC) beads. Figure 4 C-4D shows an assay showing deposition of C1q and C4 on serum exposed PS lipid beads. No deposition was observed on PC beads. Figure 4E-4F shows a competition assay demonstrating reduced deposition of C1q and C4 on serum-exposed PS lipid beads in the presence of anti-C1q neutralizing antibodies.

[0013] Figure 5 A-5D shows retinal PK / PD after anti-C1q treatment. Figure 5 A-5D shows PK / PD data showing measurable drug levels in retinal lysates from anti-C1q treated animals, and significant reductions in C1q, C1s, and C3d levels following anti-C1q treatment.

[0014] Figure 6 A-6C shows the distribution of C1q in the human GA retina: Figure 6 A-6B shows IF, which shows reduced immunoreactivity of the presynaptic marker Vglut1 ( Figure 6 A) and increased C1q labeling in the photoreceptor synaptic layer OPL ( Figure 6 B), demonstrates synaptic loss and C1q accumulation in GA retinas compared to healthy donors. Figure 6 C shows triple immunolabeling of C1q (grey), the presynaptic marker Vglut1, and a postsynaptic marker (HOMER1), confirming the colocalization of C1q with photoreceptor synapses in human GA donor retinas.

[0015] Figure 7 Human C1q binding assay is shown. Binding of Mab2-Fab, FabA, and Mab2 to human C1q in a one-sided ELISA. The bound antibody or Fab molecules were detected using enzyme-labeled anti-human Fc or anti-human κ antibodies followed by enzyme substrates. These antibodies showed comparable binding affinity to human C1q. The EC50s for Mab2-Fab, FabA, and Mab2 were 4.4, 2.5, and 4.9 ng / mL, respectively (range 34–95 pM).

[0016] Figure 8 Show, FabA inhibits classical pathway, but does not inhibit lectin and alternative complement pathway.Use the ELISA-based assay kit from Eurodiagnostica (WeislabTM) to evaluate the ability of FabA and Mab2 to inhibit classical, lectin and alternative pathways.Poles are coated with specific activators of the classical pathway (IgM), lectin pathway (mannan) or alternative pathway (lipopolysaccharide), and C5b-9 terminal complex detection antibody is used to evaluate the activation of all pathways.Inhibitory antibodies against C5 are used as positive controls.FabA and Mab2 selectively block the classical pathway with IC50≤0.3μg / mL, while anti-C5 inhibits all three pathways.

[0017] Fig. 9 Inhibition of hemolysis of IgM-coated RBCs in human serum is shown. Sheep RBCs pre-sensitized with surface-reactive polyclonal IgM antibodies were co-incubated with human serum (diluted 100-fold) at 37°C for 20-30 minutes. RBC hemolysis was quantified by measuring the release of hemoglobin and expressed as a percentage of hemolysis caused by untreated.

[0018] Fig.10 The number of damaged axons of the optic nerve of eyes treated with Mab1-Fab, Mab1 or Mab2 was shown to be reduced. By injecting 1 μl of 6 μm polystyrene beads, 1 μl of 10 μm polystyrene beads (PolybeadMicrospheres; Polysciences, Inc., Warrington, PA, USA) and 1 μl of viscoelastic solution (10 mg / mL sodium hyaluronate; Advanced Medical Optics Inc., USA) into the anterior chamber of the eye on the first day, an increase in IOP was induced in a single eye of each animal. The contralateral eye remained untouched as a control. One day before microbead injection and one week later (Day 0 and Day 7; 2 μL of 10 mg / mL antibody saline solution and saline alone were injected each time), antibodies Mab2, Mab1 and Mab1-Fab (Fab produced by enzymatic digestion of Mab1) and saline were administered intravitreally to microbead-injected eyes. Two weeks after injury, optic nerves were collected from animals (perfused with saline and 4% paraformaldehyde), post-fixed with 4% paraformaldehyde and 1% osmium, dehydrated in increasing alcohol concentrations and placed in 1% uranyl acetate / ethanol. Nerves were embedded in epoxy resin and semi-thin sections (1 μm) were cut. The total number of degenerating axons was estimated using StereoInvestigator software (MicroBrightfield, Inc, VT, USA). Scale bar = 20 μm. Both Mab1-Fab and Mab2 significantly reduced the formation of damaged axons in the optic nerve, while antibody Mab1 showed a similar trend.

[0019] Figures 11A-11D The protective effects of Mab1 antibody on photoreceptor neuron loss and retinal function in a mouse light damage model were shown. Fig.11A A 7-day mouse light damage model is shown, followed by 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 by IVT administration on day 7. Fig. 11B It was shown that Mab1 treatment resulted in a significant reduction in Tunel+ve photoreceptor cells in the outer nuclear layer of the retina compared to isotype controls. Fig. 11Cshowed that Mab1 treatment resulted in an increase in the number of photoreceptor cell rows in the outer nuclear layer compared with the isotype control. Fig.11D It was shown that Mab1 antibody treatment resulted in a significant increase in A-wave and B-wave in the electroretinogram at day 14 compared to the isotype control antibody.

[0020] Fig.12 Shown is free C1q in aqueous humor after a single IVT injection.

[0021] Figures 13A-13D Immunofluorescence (IF) data are shown. Fig. 13B Shown is reduced microgliosis in the outer plexiform layer (OPL) (also called outer synaptic layer) of the retina at day 3 after treatment. The reduction in microgliosis is associated with reduced inflammation. Fig. 13C showed remarkable preservation of photoreceptor synapses, and Fig.13D Significant retention of cell bodies was shown at day 5 after treatment.

[0022] Figures 14A-14B Measurable PK and target engagement in the retina are shown.

[0023] Fig.15A are bar graphs showing quantification of immunofluorescence images.

[0024] Fig. 15B Shown are immunofluorescence images demonstrating preservation of photoreceptor synapses (BSN marker) after treatment with a C1q inhibitor. DETAILED DESCRIPTION

[0025] Overview

[0026] 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, choroidal coloboma, fundus xanthophy, cone-rod dystrophy, and Leber congenital amaurosis) or retinal detachment.

[0027] Disclosed herein is a recombinant humanized immunoglobulin G (IgG1) antigen binding fragment (Fab) that inhibits the classical complement cascade but does not affect the lectin or alternative complement pathways. Anti-C1q Fab (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) is developed as an intravitreal (IVT) administered agent for the treatment of inherited retinal diseases (IRDs) (e.g., retinitis pigmentosa, choroidal coloboma, fundus yellow spot disease, cone-rod dystrophy and Leber congenital amaurosis) and retinal detachment. The hypervariable region derived from the murine 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 expressed as a human IgG1 Fab fragment construct (FabA). A full-length human IgG4 antibody (Mab2, i.e., an antibody comprising a heavy chain variable domain of SEQ ID NO: 8 and a light chain variable domain of SEQ ID NO: 4) containing a hypervariable region derived from Mab1 was also expressed. Mab1 and Mab2 and their Fabs (Mab1-Fab and Mab2-Fab) were used as surrogate molecules for FabA in pharmacological studies. As a monovalent Fab construct lacking Fc heavy chain constant domains 2 and 3 (CH2 and CH3), FabA cannot bind to C1q through Fc domain interactions. In addition, FabA has only one antigen binding arm and does not exhibit agonistic activity on C1q over a wide range of FabA concentrations.

[0028] The complement cascade is a key component of innate immunity and can be activated through three different pathways: the classical pathway, the lectin pathway, and the alternative complement pathway. All three pathways lead to the activation of complement component C3, ultimately leading to immune cell recruitment, inflammation, membrane lysis by the membrane attack complex, and cell death.

[0029] C1q is the initiator of the classical complement cascade and is implicated in the initiation and propagation of neurodegenerative diseases. C1q inhibition can block the initiation of the classical complement cascade and mitigate neuronal and synaptic damage by directly reducing damage to neuronal cell membranes and by reducing the inflammatory consequences of complement activation.

[0030] As measured by Biacore (<10 pM) and enzyme-linked immunosorbent assay (ELISA) (40–50 pM; Figure 7), Mab2-Fab and / or FabA exhibited high affinity binding to human C1q as measured by ELISA. Mab1 bound to the isolated globular head domain of C1q, but not to the collagen tail of C1q (as determined by ELISA). Consistent with this finding, Mab1 inhibited substrate interactions mediated by the globular head domain of C1q (IgM, C-reactive protein [CRP], and phosphatidylserine); and FabA inhibited the functional interaction of C1q with immunoglobulin M (IgM)-coated red blood cells (RBCs) (blocking hemolysis; Fig. 9 ). Antibody Mab1 specifically recognized C1q and was shown not to bind to other complement components (C3b and C5) or other C1q / tumor necrosis factor (TNF) superfamily members, including TNF and adiponectin, which are proteins that share the highest sequence identity with C1q in its globular head domain. Consistent with these results, FabA did 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 ).

[0031] definition

[0032] As used herein in the specification, "a" or "an" may mean one or more. As used herein in one or more claims, when used in conjunction with the word "comprising", the word "a" or "an" may mean one or more than one. For example, a reference to an "antibody" is a reference from one to a plurality of antibodies. As used herein, "another" may refer to at least a second or more than one.

[0033] As used herein, administration "in conjunction" with another compound or composition includes simultaneous administration and / or administration at different times. Administration in conjunction also encompasses administration as a co-formulation or administration as a separate composition, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration.

[0034] The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The term "antibody" is used in the broadest sense herein, and specifically includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two complete antibodies, antibody fragments (as long as they exhibit biological activity), and antibody derivatives.

[0035] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two % identical light chains (L) and two % identical heavy chains (H). H and VL Paired together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0036] The L chains from any vertebrate species can be divided into two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domains. Immunoglobulins can be divided into different classes or isotypes, depending on the amino acid sequence of the constant domain of their heavy chains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, whose heavy chains are named alpha ("α"), delta ("δ"), epsilon ("ε"), gamma ("γ"), and muon ("μ"), respectively. Based on relatively minor differences in CH sequence and function, the gamma and alpha classes are further divided into subclasses (isotypes), 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, for example, in Abbas et al., Cellular and Molecular Immunology, 4th ed. (WB Saunders Co., 2000).

[0037] "Full-length antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, which consist of two % identical light (L) chains and two % identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V) at one end. H ), followed by a number of constant domains. Each light chain has a variable domain at one end (V L ), and at the other end there is a constant domain; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the heavy chain variable domain. It is believed that specific amino acid residues form the interface between the light chain variable domain and the heavy chain variable domain.

[0038] An "isolated" molecule or cell is one that has been identified and separated from at least one contaminant molecule or cell with which it is normally associated in the environment in which it is produced. Preferably, the isolated molecule or cell is not associated with all components associated with the environment in which it is produced. The isolated molecule or cell is in a form different from its form or environment in nature. Thus, an isolated molecule is different from a molecule that naturally occurs in a cell; an isolated cell is different 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.

[0039] An "isolated" antibody is one that has been identified, separated, and / or recovered from a component of the environment in which it was produced (e.g., naturally or recombinantly). Preferably, an isolated polypeptide is free from association with all other contaminating components from the environment in which it was produced. Contaminating components from the environment in which it was produced (e.g., from recombinant transfected cells) are substances that typically interfere with the research, diagnostic, or therapeutic use 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 of the antibody, as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight of the antibody; (2) by use of a spinning cup sequencer to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. An isolated antibody includes the antibody in situ in a recombinant T cell, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated polypeptide or antibody will be prepared by a method that includes at least one purification step.

[0040] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy chain or light chain of an antibody. The variable domains of the heavy chain and light chain can be referred to as "V H ” and “V L These domains are usually the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding site.

[0041] The term "variable" refers to certain segments of the variable domain in an antibody that differ greatly in sequence. The V domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed over the entire span of the variable domain. In contrast, it is concentrated in three segments called hypervariable regions (HVRs) in the light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework regions (FRs). The variable domains of native heavy and light chains each contain four FR regions, which mostly adopt a β-folded configuration, connected by three HVRs, which form a loop connecting the β-sheet structure, and sometimes form a part of the β-sheet structure. The HVRs in each chain are tightly bound together by the FR region, and together with the HVRs from the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991)). The constant domains are not directly involved in the binding of antibodies to antigens, but exhibit various effector functions, such as antibody involvement in antibody-dependent cellular toxicity.

[0042] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991) (also referred to herein as Kabat 1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987) (also referred to herein as Chothia 1987); and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996) describe CDRs, where the definitions include overlaps or subsets of amino acid residues when compared to each other. However, the application of any definition to refer to the CDRs of an antibody or a grafted antibody or variants thereof is intended to be within the scope of the term as defined and used herein.

[0043] As used herein, the terms "CDR-L1", "CDR-L2" and "CDR-L3" refer to the first, second and third CDRs in the light chain variable region, respectively. As used herein, the terms "CDR-H1", "CDR-H2" and "CDR-H3" refer to the first, second and third CDRs in the heavy chain variable region, respectively. As used herein, the terms "CDR-1", "CDR-2" and "CDR-3" refer to the first, second and third CDRs in either chain variable region, respectively.

[0044] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies in the population are identical, except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies have a high degree of specificity (for a single antigenic site). In contrast to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to its specificity, monoclonal antibodies are advantageous because they are typically synthesized by hybridoma cultures and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the characteristics of an antibody obtained as a substantially homogeneous antibody population and should not be construed as requiring the antibody to be produced by any ad hoc method. For example, monoclonal antibodies used in accordance with the present disclosure can be prepared by a variety of techniques, including, for example, the hybridoma method (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, 2nd ed. 1988); Hammerling et al., Monoclonal Antibodies and T-Cell Immunofluorescence ... Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage display technology (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et 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 having partial or complete human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, 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. Patents 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 Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0045] The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably to refer to an antibody in substantially complete form, as opposed to an antibody fragment or antibody derivative. Specifically, whole antibodies include those antibodies having heavy and light chains that include an Fc region. The constant region may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, a complete antibody may have one or more effector functions.

[0046] An "antibody fragment" or "antigen-binding fragment" or "functional fragment" of an antibody comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of an intact antibody, or the F region of an antibody that retains or has modified FcR binding ability. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; and linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)). Other examples of antibody fragments include antibody derivatives, such as single-chain antibody molecules, monovalent antibodies, and multispecific antibodies formed from antibody fragments.

[0047] An "antibody derivative" is any construct comprising the antigen-binding region of an antibody. Examples of antibody derivatives include single-chain antibody molecules, monovalent antibodies, and multispecific antibodies formed from antibody fragments.

[0048] Papain digestion of antibodies produces two % identical antigen-binding fragments, called "Fab" fragments, and a residual "Fc" fragment, a name that reflects the ability to crystallize readily. The Fab fragment consists of an intact L chain and the variable region domain (V H ) and the first constant domain of a heavy chain (C H 1) Composition. Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment that is roughly equivalent to two disulfide-linked Fab fragments with different antigen binding activities and is still able to cross-link antigen. The Fab' fragment differs from the Fab fragment in that the C H The carboxyl terminus of the constant domain has several additional residues, including one or more cysteines from the antibody hinge region. Fab' in which the cysteine ​​residues of the constant domains have free thiol groups is referred to herein as Fab'-SH. F(ab')2 antibody fragments were initially produced in the form of Fab' fragment pairs that had hinge cysteines between the Fab' fragments. Other chemical couplings of antibody fragments are also known.

[0049] The Fc fragment comprises the carboxyl terminal portions of two H chains held together by disulfides. The effector functions of an antibody are determined by the sequence of the Fc region, which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0050] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be different, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (according to the EU numbering system, residue 447) in the Fc region can be removed, for example, during production or purification of antibodies or by recombinant engineering of the nucleic acid encoding the heavy chain of an antibody. Therefore, the composition of a complete antibody can include an antibody population in which all K447 residues are removed, an antibody population in which no K447 residue is removed, and an antibody population with and without an antibody mixture containing K447 residues. The native sequence Fc region suitable for antibodies of the present disclosure includes human IgG1, IgG2, IgG3, and IgG4.

[0051] A "native sequence Fc region" comprises an amino acid sequence that is % identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include native sequence human IgG1 Fc regions (non-A and A allotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, as well as naturally occurring variants thereof.

[0052] A "variant Fc region" comprises an amino acid sequence that is distinguished from a native sequence Fc region amino acid sequence by at least one amino acid modification (preferably one or more amino acid substitutions). Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, for example, there are about one to about ten amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide, preferably about one to about five amino acid substitutions. The variant Fc region herein will preferably have at least about 80% homology with the native sequence Fc region and / or with the Fc region of a parent polypeptide, most preferably at least about 90% homology therewith, and more preferably at least about 95% homology therewith.

[0053] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is an FcR that binds to IgG antibodies (gamma receptors) and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternative splice forms of these receptors, FcγRII receptors including FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif ("ITAM") in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif ("ITIM") in its cytoplasmic domain. (See, e.g., M. 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 deHaas et al., J. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs (including those to be identified in the future) are encompassed by the term "FcR". FcRs can also increase the serum half-life of antibodies.

[0054] The binding and serum half-life of a human FcRn high affinity binding polypeptide in vivo to FcRn can be determined, for example, in transgenic mice expressing human FcRn or transfected human cell lines or in primates to which polypeptides having variant Fc regions are administered. WO 2004 / 42072 (Presta) describes antibody variants with enhanced or reduced binding to FcRs. See also, for example, Shields et al., J. Biol. Chem. 9 (2): 6591-6604 (2001).

[0055] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight non-covalent association. From the folding of these two domains, six hypervariable loops (3 loops each in the H chain and L chain) are derived, which provide amino acid residues for antigen binding and give the antibody antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind to an antigen, although its affinity is lower than that of the complete binding site.

[0056] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising the VH and VL antibody domains linked into a single polypeptide chain. Preferably, the sFv polypeptide also comprises a V H With V L A polypeptide linker between domains that enables sFv to form the structure required for antigen binding. For a review of sFv, see Plückthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0057] The term "diabody" refers to a H With V L Small antibody fragments prepared by constructing sFv fragments (see previous paragraph) with short linkers (about 5-10 residues) between the domains, so that interchain pairing of the V domains is achieved rather than intrachain pairing, thereby generating bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the V domains of the two antibodies are H and V L The domains are present on different polypeptide chains. Diabodies are described in detail in, for example, 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).

[0058] As used herein, "chimeric antibody" refers to an antibody (immunoglobulin) in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as long as it exhibits the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Nat'l Acad. Sci. USA, 81:6851-55 (1984)). The chimeric antibodies of interest herein include Antibodies, wherein the antigen binding region of the antibody is derived from an antibody produced by, for example, immunizing a macaque with an antigen of interest. As used herein, "humanized antibodies" are a subset of "chimeric antibodies."

[0059] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing minimal sequences derived from non-human immunoglobulins. In some embodiments, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the HVR of the receptor are replaced by residues from the HVR of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) with the desired specificity, affinity, and / or ability. In some cases, the FR residues of a human immunoglobulin are replaced by corresponding non-human residues. In addition, a humanized antibody may be included in a receptor antibody or in a residue not found in a donor antibody. These modifications may be performed to further improve the performance of the antibody, such as binding affinity. In general, a humanized antibody will include substantially all of at least one, usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, but the FR region may include one or more individual FR residue substitutions, which may improve antibody performance, such as binding affinity, isomerization, immunogenicity, etc. The number of these amino acid substitutions in the FR is usually no more than 6 in the H chain and no more than 3 in the L chain. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional details, see, e.g., Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

[0060] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or an antibody prepared using any of the techniques disclosed herein for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). Also useful for preparing human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1): 86-95 (1991). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled, such as an immunized xenogeneic mouse (refer to XENOMOUSE TM For human antibodies generated by human B cell hybridoma technology, see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584). For human antibodies generated by human B cell hybridoma technology, see, e.g., Li et al., Proc. Nat'l Acad. Sci. USA, 103:3557-3562 (2006).

[0061] The term "hypervariable region", "HVR" or "HV" as used herein refers to the region of the antibody variable domain whose sequence is highly variable and / or forms a structurally defined loop. Typically, an antibody comprises six HVRs, three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In natural antibodies, H3 and L3 show the greatest diversity among the six HVRs, and H3 in particular is considered to play a unique role in conferring good specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, 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, eg, Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0062] Many HVR descriptions are being used and are included in this article. HVR as Kabat complementary determining region (CDR) is based on the variability of sequence, and is the most commonly used (Kabat et al., supra). Chothia refers to the position of the structural loop (Chothia and Lesk J.Mol.Biol.196:901-917 (1987)). AbM HVR represents a compromise between Kabat CDR and Chothia structural loops, and is used by the AbM antibody modeling software of Oxford Molecular. "Contact" HVR is based on the analysis of available complex crystal structures. The residues from each of these HVRs are as follows.

[0063]

[0064] HVRs may include the following "extended HVRs": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, 26-35 (H1), 50-65 or 49-65 (preferred embodiments) (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these extended HVR definitions, the variable domain residues are numbered according to Kabat et al., supra.

[0065] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as herein defined.

[0066] The phrase "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variants thereof refer to the numbering system used for the heavy chain variable domain or light chain variable domain of the antibody compilation 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 or insertion of a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). For a given antibody, the Kabat numbering of the residues can be determined by aligning the homologous region of the antibody sequence with the "standard" Kabat numbering sequence.

[0067] When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., Sequences of Immunological Interest. 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., Kabat et al., EU index reported in supra). "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise specified herein, reference to residue numbering in antibody variable domains refers to residue numbering by the Kabat numbering system. Unless otherwise specified herein, reference to residue numbering in antibody constant domains refers to residue numbering by the EU numbering system (see, e.g., U.S. Patent Publication No. 2010-280227).

[0068] As used herein, "acceptor human framework" is a framework comprising an amino acid sequence of a VL or VH framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise an amino acid sequence that is % identical to it, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. When pre-existing amino acid changes are present in VH, it is preferred that these changes occur only at three, two or one positions in positions 71H, 73H and 78H; for example, the amino acid residues at those positions may be 71A, 73T and / or 78A. In some embodiments, the VL acceptor human framework sequence is % identical to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0069] "Human consensus framework" is a framework that represents the amino acid residues that are most commonly present in a selection of human immunoglobulin VL or VH framework sequences. Typically, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is a subgroup such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD (1991). Examples include that for VL, the subgroup may be a subgroup κI, κII, κIII or κIV such as Kabat et al., supra. In addition, for VH, the subgroup may be a subgroup I, subgroup II or subgroup III such as Kabat et al., supra.

[0070] "Amino acid modification" at a specific position refers to the substitution or deletion of a specific residue, or the insertion of at least one amino acid residue near a specific residue. Insertion "adjacent" a specific residue refers to insertion within one to two residues thereof. Insertion can be at the N-terminus or C-terminus of a specific residue. The preferred amino acid modification herein is substitution.

[0071] An "affinity matured" antibody refers to an antibody that has one or more changes in one or more of its HVRs that result in an improved affinity of the antibody for the antigen compared to a parent antibody that does not have those changes. In some embodiments, affinity matured antibodies have nanomolar or even picomolar affinities for the target antigen. Affinity matured antibodies are produced by methods known in the art. For example, Marks et al., Bio / Technology 10:779-783 (1992) describe affinity maturation by VH and VL domain shuffling. Random mutagenesis of HVR and / or framework residues is described, e.g., 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).

[0072] As used herein, the term "specific recognition" or "specific binding" refers to a measurable and repeatable interaction, such as attraction or binding between a target and an antibody, which determines the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically or preferentially binds to a target or epitope is an antibody that binds to a target or epitope with greater affinity, avidity, greater ease, and / or greater duration than it binds to other targets or other epitopes of a target. It is also understood that, for example, an antibody (or portion) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (but it may include) exclusive binding. An antibody that specifically binds to a target may have at least about 10 3 M -1 or 10 4 M -1 , sometimes about 10 5 M -1 or 10 6 M -1 , in other cases about 10 6 M -1 or 10 7 M -1 , about 10 8 M -1 Up to 10 9 M -1 or about 10 10 M -1 Up to 10 11 M -1or higher association constant. A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select monoclonal antibodies that specifically immunoreact with a protein. For a description of immunoassay formats and conditions that can be used to determine specific immune responses, see, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0073] As used herein, "identity" means that at any particular position of the aligned sequences, the amino acid residues between the sequences are % identical. As used herein, "similarity" indicates that at any particular position of the aligned sequences, the amino acid residues between the sequences are of similar type. For example, leucine can replace isoleucine or valine. Other amino acids that can often be substituted for each other include, but are not limited to:

[0074] - Phenylalanine, tyrosine and tryptophan (amino acids with aromatic side chains);

[0075] - lysine, arginine and histidine (amino acids with basic side chains);

[0076] - Aspartic acid and glutamic acid (amino acids with acidic side chains);

[0077] - Asparagine and glutamine (amino acids with amide side chains); and

[0078] - Cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0079] The degree of identity and similarity can be readily calculated (see, e.g., Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG, ed., Humana Press, New Jersey, 1994; Sequence Analyses is in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., M Stockton Press, New York, 1991).

[0080] As used herein, an "interaction" between a complement protein and a second protein encompasses, but is not limited to, protein-protein interactions, physical interactions, chemical interactions, binding, covalent binding, and ionic binding. As used herein, an antibody "inhibits" an interaction between two proteins when the antibody disrupts, reduces, or completely eliminates the interaction between the two proteins. When an antibody or fragment thereof of the present disclosure binds to one of the two proteins, the antibody or fragment thereof "inhibits" an interaction between the two proteins.

[0081] A "blocking" antibody, "antagonist" antibody, "inhibitory" antibody, or "neutralizing" antibody is an antibody that inhibits or reduces one or more biological activities of the antigen to which it binds, such as interaction with one or more proteins. In some embodiments, a blocking antibody, antagonist antibody, inhibitory antibody, or "neutralizing" antibody substantially or completely inhibits one or more biological activities or interactions of the antigen.

[0082] The term "inhibitor" refers to a compound that can inhibit the biological function of a target biomolecule (e.g., mRNA or protein) by reducing 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 inhibits the biological response of the receptor. The term "inhibitor" may also refer to an "antagonist."

[0083] Antibody "effector functions" refer to those 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.

[0084] As used herein, the term "affinity" refers to the equilibrium constant that two reagents (e.g., antibody and antigen) are reversibly combined, and is expressed as a dissociation constant (KD). Affinity can be at least 1 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, or at least 1,000 times or more than the affinity of the antibody to an unrelated amino acid sequence. The affinity of the antibody to a target protein can be, for example, about 100 nanomoles (nM) to about 0.1 nM, about 100 nM to about 1 picomoles (pM) or about 100 nM to about 1 femtomoles (fM) or higher. As used herein, the term "affinity" refers to the resistance of a complex of two or more agents to dissociate upon dilution. With respect to antibodies and / or antigen-binding fragments, the terms "immunoreactivity" and "preferential binding" are used interchangeably herein.

[0085] The term "binding" refers to a direct association between two molecules due to, for example, 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 an affinity of at least about 10 -7 M or larger, such as 5×10 -7 M, 10 -8 M, 5×10 -8 M and greater. "Non-specific binding" refers to an affinity less than about 10 -7 M, for example, 10 -6 M, 10 -5 M, 10 -4 M and other affinity binding.

[0086] The term "k on ” refers to the rate constant for the association of antibody with antigen.

[0087] The term "k off ” refers to the rate constant for the dissociation of an antibody from the antibody / antigen complex.

[0088] The term "K D ” refers to the equilibrium dissociation constant of the antibody-antigen interaction.

[0089] As used herein, "percentage (%) of amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after alignment of the sequences and introduction of gaps (if necessary) to achieve the maximum percentage of sequence identity, and does not take into account any conservative substitutions as part of the sequence identity. Alignment for determination of percentage of amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms known in the art needed to achieve maximal alignment over the full length of the sequences being compared.

[0090] "Biological sample" covers a variety of sample types obtained from an individual, and can be used for diagnosis or monitoring determination. The definition covers liquid samples of blood and other biological sources, solid tissue samples (such as biopsy specimens or tissue cultures or cells derived therefrom) and their offspring. The definition also includes samples processed in any way after acquisition, such as by treating with reagents, dissolving or enriching certain components, such as polynucleotides. The term "biological sample" covers 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, eye fluid, synovial fluid, blood components (such as plasma and serum), etc. The term "biological sample" also includes solid tissue samples, tissue culture samples and cell samples.

[0091] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminating nucleic acid molecule with which it is normally associated in the environment in which it is produced. Preferably, the isolated nucleic acid is not associated 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 different from the form or environment in which they are found in nature. Thus, the isolated nucleic acid molecules are different from the nucleic acids encoding any polypeptides and antibodies that are naturally present in cells.

[0092] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting other nucleic acids connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA to which an additional DNA segment can be connected. Another type of vector is a bacteriophage vector. Another type of vector is a viral vector, in which an additional DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". Typically, expression vectors useful in recombinant DNA technology are generally in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms.

[0093] "Polynucleotide" or "nucleic acid" used interchangeably herein refers to a polymer of nucleotides of any length, and includes 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. The polynucleotide can contain modified nucleotides, such as methylated nucleotides and their analogs. If present, the nucleotide structure can be modified before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide can contain modifications performed after synthesis, such as conjugation with a label. Other types of modifications include, for example, "caps" (replacing one or more naturally occurring nucleotides with analogs); internucleotide modifications, such as those with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.); those containing side groups, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.); those with intercalators (e.g., acridine, psoralen, etc.); those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.); those containing alkylating agents; those with modified bonds (e.g., alpha anomeric nucleic acids, etc.); and unmodified forms of polynucleotides. In addition, any hydroxyl groups typically present in sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional connections to additional nucleotides, or may be conjugated to solid or semisolid supports. 5' and 3' terminal OH can be phosphorylated or partially replaced by amine or organic end-capping groups of 1 to 20 carbon atoms. Other hydroxyls can also be derived into standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose 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, furanose, sedoheptulose (sedoheptulose), acyclic analogs and alkaline nucleoside analogs (such as methyl riboside). One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("methyl acetal"), wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl group optionally containing an ether (-O-) linkage. Not all linkages in a polynucleotide need be identical.The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0094] "Host cell" includes a single cell or cell culture that can be or has been a recipient of a vector for incorporating a polynucleotide insert. Host cells include the progeny of a single host cell, and the progeny are not necessarily completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutation. Host cells include cells transfected in vivo with a polynucleotide of the disclosure.

[0095] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is nontoxic to cells or mammals exposed thereto at the dosages and concentrations used. Typically, a physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, 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 dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN TM , polyethylene glycol (PEG) and PLURONICS TM .

[0096] The terms "prevent" and "prevent" are art-recognized and when used in connection with a condition such as an inherited retinal disease (IRD) (e.g., retinitis pigmentosa / rod-cone dystrophy, choroidal coloboma, fundus xanthophy, cone-rod dystrophy, Leber congenital amaurosis, X-linked RP, and Usher syndrome) or retinal detachment or related symptoms, are relative to an untreated patient.

[0097] The term "subject" as used herein refers to a living mammal and can be used interchangeably with the term "patient". Examples of mammals include, but are not limited to, any member of the class of mammals: humans; non-human primates, such as chimpanzees and other apes and monkey species; livestock, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs and cats; laboratory animals, including rodents, such as rats, mice and guinea pigs, etc. The terms do not denote a particular age or sex.

[0098] As used herein, the terms "treating" or "treatment" include alleviating a disorder, arresting a disorder, or reversing the symptoms, clinical signs, or underlying pathology of a disorder, to stabilize or improve a subject's disorder, or to reduce the likelihood that a subject's disorder will worsen, as if the subject were not receiving treatment.

[0099] "Recovery" refers to the act of returning to a normal or healthy state. Recovery can be partial (e.g., when the subject returns to a condition less than normal or healthy) or complete (e.g., when the subject returns to a condition that is the same or nearly the same as normal or healthy). An example of a normal or healthy state is the vision of a patient before retinal detachment.

[0100] "Improving vision" refers to the act of enhancing the ability or state of being able to see relative to pre-treatment, including improving visual acuity, sensitivity and / or visual field range.

[0101] The term "therapeutically effective amount" of a compound with respect to the subject treatment methods refers to an amount of the compound in a formulation that, when administered as part of a desired dosing regimen (to a mammal, preferably a human), alleviates symptoms, ameliorates symptoms, or slows the onset of a disease condition, according to clinically acceptable standards for the condition or disorder to be treated or for cosmetic purposes, e.g., at a reasonable benefit / risk ratio applicable to any drug treatment. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual.

[0102] As used herein, an individual who is "at risk" of developing a particular disease, disorder, or condition may or may not have detectable disease or disease symptoms, and may or may not have detectable disease or disease symptoms prior to the methods of treatment described herein. "At risk" means that the individual has one or more risk factors, which are measurable parameters associated with the development of a particular disease, disorder, or condition, as known in the art. An individual who has one or more of these risk factors has a higher likelihood of developing a particular disease, disorder, or condition than an individual who does not have one or more of these risk factors.

[0103] "Chronic" administration refers to administration in a continuous rather than acute manner, thereby maintaining the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration refers to treatment that is not continuously administered without interruption, but is cyclical / periodic in nature.

[0104] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of the present invention, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (ed. FMAusubel et al. (2003)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (edited by MJMacPherson, BD Hames and GRTaylor (1995)), Harlow and Lane (edited (1988)) Antibodies, ALaboratory Manual, and Animal Cell Culture (edited by RI Freshney, (1987)); Oligonucleotide Synthesis (edited by MJGait, 1984); Methods in Molecular Biology, Humana Press; CellBiology: A Laboratory Notebook (edited by JECellis, 1998) Academic Press; Animal CellCulture (RI Freshney), ed., 1987); Introduction to Cell and Tissue Culture (JPMather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JBGffiths and DG Newell, ed., 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (DMWeir and CCBlackwell (ed.); Gene TransferVectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The PolymeraseChain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 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 (edited by D. Catty, IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory The widely used methods described in The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 1993).

[0105] Antibody

[0106] All sequences mentioned in the present disclosure are incorporated by reference into U.S. Patent Application No. 14 / 933,517, U.S. Patent Application No. 14 / 890,811, U.S. Patent No. 8,877,197, U.S. Patent No. 9,708,394, U.S. Patent Application No. 15 / 360,549, U.S. Patent No. 9,562,106, U.S. Patent No. 10,450,382, U.S. Patent No. 10,457,745, International Patent Application No. PCT / US2018 / 022462, and the antibodies and related compositions disclosed in each of these patents are hereby incorporated by reference.

[0107] Full-length antibodies can be prepared by using recombinant DNA engineering techniques. Such engineered versions include versions produced, for example, by inserting, deleting or changing the variable region of a natural antibody in the amino acid sequence of a natural antibody. Specific examples of this type include those engineered variable region domains containing at least one CDR and optionally one or more framework amino acids from an antibody and the remainder of the variable region domain from a second antibody. The DNA encoding the antibody can be prepared by all parts except the required part in the DNA encoding the full-length antibody by deletion. The DNA encoding the chimeric antibody can be prepared by recombining the DNA encoding the variable region of the human constant region substantially or specifically and encoding the variable region of the variable region sequence of the mammals other than humans substantially or specifically. The DNA encoding the humanized antibody can be prepared by recombining the DNA encoding the constant region and the variable region except the complementary determining region (CDR) of the corresponding human antibody region substantially or specifically and encoding the DNA of the CDR of the mammals other than humans substantially or specifically.

[0108] Suitable sources of DNA molecules encoding antibodies include cells expressing full-length antibodies, such as hybridomas. For example, antibodies can be isolated from host cells expressing expression vectors encoding the heavy and / or light chains of the antibodies.

[0109] Antibody fragments, including but not limited to Fab fragments and / or antibody derivatives, can also be prepared using recombinant DNA engineering techniques, which involve the manipulation and re-expression of DNA encoding the variable and constant regions of antibodies. Standard molecular biology techniques can be used to modify, add or delete more amino acids or domains as needed. The terms "variable" and "constant" regions as used herein still encompass any changes to the variable or constant regions. In some cases, by modifying the C H A stop codon was introduced immediately after the codon for the interchain cysteine ​​of 1, and PCR was used to generate antibody fragments such that C H The translation of the 1 domain is terminated at the interchain cysteine. Methods for designing suitable PCR primers are well known in the art, and antibody C H The sequence of the 1 domain is readily available. In some embodiments, a stop codon can be introduced using site-directed mutagenesis techniques.

[0110] The antibodies of the present disclosure can be derived from any antibody isotype ("class"), including, for example, IgG, IgM, IgA, IgD and IgE and subclasses thereof, including, for example, IgG1, IgG2, IgG3 and IgG4. In certain preferred embodiments, the heavy and light chains of the antibodies are from IgG. The heavy and / or light chains of the antibodies can be from mouse IgG or human IgG. In certain other preferred embodiments, the heavy and / or light chains of the antibodies are from human IgG1. In yet other preferred embodiments, the heavy and / or light chains of the antibodies are from human IgG4.

[0111] The antibodies disclosed herein can bind to and inhibit the biological activity of C1q, C1r or C1s. For example, (1) binding of C1q to autoantibodies, (2) binding of C1q to C1r, (3) binding of C1q to C1s, (4) binding of C1q to phosphatidylserine, (5) binding of C1q to pentraxin-3, (6) binding of C1q to C-reactive protein (CRP), (7) binding of C1q to globular C1q receptor (gC1qR), (8) binding of C1q to complement receptor 1 (CR1), (9) binding of C1q to β-amyloid protein, or (10) binding of C1q to calreticulin. In other embodiments, the biological activity of C1q is (1) activation of the classical complement activation pathway, (2) reduction in lysis and / or reduction in C3 deposition, (3) activation of antibodies and complement dependent cytotoxicity, (4) CH50 hemolysis, (5) reduction in erythrocyte lysis, (6) reduction in erythrocyte phagocytosis, (7) reduction in dendritic cell infiltration, (8) inhibition of complement-mediated erythrocyte lysis, (9) reduction in lymphocyte infiltration, (10) reduction in macrophage infiltration, (11) reduction in antibody deposition, (12) Reduction in neutrophil infiltration, (13) Reduction in platelet phagocytosis, (14) Reduction in platelet lysis, (15) Improvement in transplant graft survival, (16) Reduction in macrophage-mediated phagocytosis, (17) Reduction in autoantibody-mediated complement activation, (18) Reduction in red blood cell destruction caused by transfusion reactions, (19) Reduction in red blood cell lysis caused by alloantibodies, (20) Reduction in hemolysis caused by transfusion reactions, (21) Reduction in alloantibody-mediated platelet lysis, (22) Improvement in anemia , (23) reduction in eosinophilia, (24) reduction in C3 deposition on red blood cells (e.g., reduction in deposition of C3b, iC3b, etc. on RBCs), (25) reduction in C3 deposition on platelets (e.g., reduction in deposition of C3b, iC3b, etc. on platelets), (26) reduction in anaphylatoxin production, (27) reduction in autoantibody-mediated blister formation, (28) reduction in autoantibody-induced erythema, (29) reduction in red blood cell destruction caused by transfusion reactions, (30) reduction in blood transfusion reactions caused by transfusion reactions reduction in platelet lysis, (31) reduction in mast cell activation, (32) reduction in mast cell histamine release, (33) reduction in vascular permeability, (34) reduction in complement deposition on the transplant graft endothelium, (35) B cell antibody production, (36) dendritic cell maturation, (37) T cell proliferation, (38) cytokine production, (39) microglial activation, (40) Arthritis reaction, (41) reduction in anaphylatoxin production in the transplant graft endothelium, or (42) activation of complement receptor 3 (CR3 / C3) expressing cells.

[0112] In some embodiments, CH50 hemolysis includes human, mouse and / or rat CH50 hemolysis. In some embodiments, the antibody is capable of neutralizing at least about 50% to at least about 95% of CH50 hemolysis. In some embodiments, the antibody is capable of neutralizing 50%, 60%, 70%, 80%, 90% or 100% of CH50 hemolysis. The antibody is also capable of neutralizing at least 50% of CH50 hemolysis at a dose of less than 150 ng / ml, less than 100 ng / ml, less than 50 ng / ml or less than 20 ng / ml.

[0113] Other in vitro assays for measuring complement activity include ELISA assays for measuring cleavage products of complement components or complexes formed during complement activation. Complement activation through the classical pathway can be measured by tracking the levels of C4d and C4 in serum. Activation of the alternative pathway can be measured in an ELISA by assessing the levels of circulating Bb or C3bBbP complexes. In vitro antibody-mediated complement activation assays can also be used to assess inhibition of C3a production.

[0114] The antibodies of the present disclosure may be monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, human antibodies, chimeric antibodies, multispecific antibodies, antibody fragments thereof, or derivatives thereof. In some embodiments, the antibody is a humanized antibody.

[0115] The antibodies of the present disclosure may also be antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies or single-chain antibody molecules. In some embodiments, the antibody fragment is a Fab fragment.

[0116] In some embodiments, the antibody is a human monoclonal antibody that can be prepared, expressed, produced, or isolated by recombinant means, such as (a) an antibody isolated from an animal (e.g., mouse) that is transgenic or cross-chromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) an antibody isolated from a host cell transformed to express the antibody (e.g., from a transfectoma), (c) an antibody isolated from a recombinant, combinatorial human antibody library, and (d) an antibody prepared, expressed, produced, or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions from human germline and / or non-germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when a transgenic animal for human Ig sequences is used, in vivo somatic mutagenesis), and thus the V H and V L The amino acid sequence of the region is such a sequence, which, although derived from human germline V H and V LSequences that are related to, but may not naturally exist within the human antibody germline repertoire in vivo.

[0117] In some embodiments, the antibody is a humanized and / or chimeric monoclonal antibody, which can be produced by immunizing rodents (e.g., mice, rats, hamsters, and guinea pigs) with (1) natural complement components (e.g., C1q) derived from enzymatic digestion of purified complement components from human plasma or serum, or (2) recombinant complement components expressed by eukaryotic or prokaryotic systems, or fragments thereof. Other animals can be used for immunization, such as non-human primates, transgenic mice expressing human immunoglobulins, and severe combined immunodeficient (SCID) mice transplanted with human B lymphocytes.

[0118] Polyclonal and monoclonal antibodies are naturally produced as immunoglobulin (Ig) molecules by the immune system in response to pathogens. The IgG1 molecule of about 150 kDa is the major form in human serum at a concentration of 8 mg / ml and is composed of two identical heavy chains of about 50 kDa and two identical light chains of about 25 kDa.

[0119] Hybridomas can be produced by conventional procedures by fusing B lymphocytes from immunized animals with myeloma cells. In addition, anti-C1q antibodies can be produced by screening recombinant single-chain Fv or Fab libraries from human B lymphocytes in a phage display system. The specificity of MAbs for human C1q can be tested by enzyme-linked immunosorbent assay (ELISA), protein immunoblotting (Western immunoblotting) or other immunochemical techniques.

[0120] The inhibitory activity of antibodies identified during the screening process on complement activation can be assessed by hemolytic assay using unsensitized rabbit or guinea pig RBCs for the alternative complement pathway or sensitized chicken or sheep RBCs for the classical complement pathway. Those hybridomas that exhibit inhibitory activity specific for the classical complement pathway are cloned by limiting dilution. Antibodies are purified by the above assays for characterization of specificity for human CIq.

[0121] Anti-complement C1q antibody

[0122] The anti-C1q antibodies disclosed herein are potent inhibitors of C1q and can be administered for any period of time to provide sustained inhibition of C1q function and then optionally discontinued to allow restoration of normal C1q function when its activity may be important. Results obtained in animal studies with the anti-C1q antibodies disclosed herein can be readily carried into the clinic with humanized or human antibodies and fragments and / or derivatives thereof.

[0123] C1q is a 460 kDa polymeric protein 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).

[0124] The antibodies disclosed herein specifically recognize complement factor C1q and / or C1q in the C1 complex of the classical complement activation pathway. The binding complement factor can be derived from any organism with a complement system without limitation, including any mammalian organism, such as humans, mice, rats, rabbits, monkeys, dogs, cats, cows, horses, camels, sheep, goats or pigs.

[0125] As used herein, "C1 complex" refers to a protein complex that may include, but is not limited to, one C1q protein, two C1r proteins, and two C1s proteins (e.g., 2 s 2 ).

[0126] The anti-C1q antibodies disclosed herein can inhibit C1 complex formation.

[0127] As used herein, "complement factor C1q" refers to both the wild-type sequence and naturally occurring variant sequences.

[0128] A non-limiting example of a complement factor C1q recognized by the antibodies of the present disclosure is human C1q, comprising three polypeptide chains A, B and C:

[0129] C1q, chain A (homo sapiens), accession number protein

[0130] Database: NP_057075.1; GenBank ID: NM_015991:

[0131] >gi|7705753|ref|NP_057075.1|Component C1q

[0132] Subunit A precursor [Homo sapiens]

[0133] (SEQ ID NO:1)

[0134] MEGPRGWLVLCVLAISLASMVTEDLCCRAPDGKKGEAGRPGRRGRPGLKGEQGEPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPGNIKDQPRPAFSAIRRN PPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFTFQVLSQWEICLSIVSSSSRGQVRRSLGFCDTTNKGLFQVVSGGMVLQLQQGDQVWVEKDPKKGHIYQGSEADSVFSGFLIFPSA.

[0135] C1q, chain B (Homo sapiens), accession number protein

[0136] Database: NP_000482.3; GenBank ID: NM_000491.3:

[0137] >gi|87298828|ref|NP_000482.3|Component C1q

[0138] Subcomponent subunit B precursor [Homo sapiens]

[0139] (SEQ ID NO:2)

[0140] MMMKIPWGSIPVLMLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTPGIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGPKGESGDYKATQKIAFSAT RTINVPLRRDQTIRFDHVITNMNNNYEPRSGKFTCKVPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVLKLEQGENVFLQATDKNSLLGMEGANSIFSGFLLFPDMEA.

[0141] C1q, chain C (Homo sapiens), accession number protein

[0142] Database: NP_001107573.1; GenBank ID:

[0143] NM_001114101.1:

[0144] >gi|166235903|ref|NP_001107573.1|Component C1q

[0145] Subunit C precursor [Homo sapiens]

[0146] (SEQ ID NO:3)

[0147] MDVGPPSSLPHLGLKLLLLLLLLPLRGQANTGCYGIPGMPGLPGAPGKDGYDGLPPGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGIPGEPGEEGRYKQKFQSVFTVTRQTHQPPAPNSLIRFNAVLTNPQGDYD

[0148] TSTGKFTCKVPGLYYFVYHASHTANLCVLLYRSGVKVVTFCGHTSKTNQVNSGGVLLRLQVGEEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD.

[0149] Thus, the anti-C1q antibodies of the present disclosure can bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of the C1q protein. In some embodiments, the anti-C1q antibodies of the present disclosure bind to polypeptide chain A, polypeptide chain B, and / or polypeptide chain C of human C1q or a homolog thereof, such as mouse, rat, rabbit, monkey, dog, cat, cow, horse, camel, sheep, goat, or pig C1q. In some embodiments, the anti-C1q antibody is a human antibody, a humanized antibody, a chimeric antibody, or a fragment or derivative thereof. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody fragment, such as a Fab fragment.

[0150] All sequences mentioned in the following twenty paragraphs are incorporated by reference into US Patent No. 9,708,394, the antibodies and related compositions disclosed therein are incorporated herein by reference.

[0151] Light chain and heavy chain variable domain sequences of antibody M1 (Mab1)

[0152] Using standard techniques, the nucleic acid and amino acid sequences encoding the light chain variable domain and heavy chain variable domain of antibody M1 were determined. The amino acid sequence of the light chain variable domain of antibody M1 is:

[0153]

[0154] The hypervariable region (HVR) of the light chain variable domain is marked in bold and underlined. In some embodiments, the HVR-L1 of the M1 light chain variable domain has the sequence RASKSINKYLA (SEQ ID NO: 5), the HVR-L2 of the M1 light chain variable domain has the sequence SGSTLQS (SEQ ID NO: 6), and the HVR-L3 of the M1 light chain variable domain has the sequence QQHNEYPLT (SEQ ID NO: 7).

[0155] The amino acid sequence of the heavy chain variable domain of antibody M1 is:

[0156]

[0157] The hypervariable region (HVR) of the heavy chain variable domain is marked in bold and underlined. In some embodiments, the HVR-H1 of the M1 heavy chain variable domain has the sequence GYHFTSYWMH (SEQ ID NO: 9), the HVR-H2 of the M1 heavy chain variable domain has the sequence VIHPNSGSINYNEKFES (SEQ ID NO: 10), and the HVR-H3 of the M1 heavy chain variable domain has the sequence ERDSTEVLPMDY (SEQ ID NO: 11).

[0158] The nucleic acid sequence encoding the light chain variable domain was determined to be:

[0159] GATGTCCAGATAACCCAGTCTCCATCTTATCTTGCTGCATCTCCTGGAAACCATTACTATTAATTGCAGGGCAAGTAAGAGCATTAACAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAGCTTCTTATCTACTCTGGATCCACTTTG CAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGTAGCCTGGAGCCTGAAGATTTTGCAATGTATTACTGTCAACAACATAATGAATACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA(SEQ ID NO:12).

[0160] The nucleic acid sequence encoding the heavy chain variable domain was determined to be:

[0161] CAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTAAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGTCTTCTGGCTACCATTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAGTGATTCATCCTAATAGTGGTAGTATTAACTACAAT GAGAAGTTCGAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCGGCGGTCTATTATTGTGCAGGAGAGAGAGATTCTACGGAGGTTCTCCCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ IDNO:13).

[0162] Mab1-Fab is the Fab of the Mab1 (M1) antibody.

[0163] Mab3 is a murine anti-Clq antibody derived from the Mab 1 antibody and optimized for murine experiments, and Mab3-Fab is the Fab of the Mab3 antibody.

[0164] Material preservation

[0165] The following materials have been deposited under the Budapest Treaty with the American Type Culture Collection, ATCC Patent Depository, 10801 University Blvd., Manassas, Va. 20110-2209, USA (ATCC):

[0166]

[0167]

[0168] The hybridoma cell line producing the M1 antibody (Mouse Hybridoma C1qM1 7788-1(M)051613) has been deposited with the ATCC under conditions that ensure availability of culture during the pendency of a patent application for a period of 30 years, or 5 years after the most recent request, or the effective life of the patent, whichever is longer. If the deposit becomes unavailable during that period, the deposit will be replaced. The deposit is available as required by foreign patent laws in countries in which the counterpart or progeny of the subject application is filed. It should be understood, however, that the availability of the deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

[0169] Disclosed herein are methods of administering an anti-C1q antibody comprising a light chain variable domain and a heavy chain variable domain. The antibody may bind at least to 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 the monoclonal antibody M1 produced by the hybridoma cell line deposited with accession number PTA-120399. The heavy chain variable domain comprises HVR-H1, HVR-H2, and HVR-H3 of the monoclonal antibody M1 produced by the hybridoma cell line deposited with ATCC accession number PTA-120399.

[0170] In some embodiments, the amino acid sequences of the light chain variable domain and the heavy chain variable domain comprise one or more of SEQ ID NO: 5 of HVR-L1, SEQ ID NO: 6 of HVR-L2, SEQ ID NO: 7 of HVR-L3, SEQ ID NO: 9 of HVR-H1, SEQ ID NO: 10 of HVR-H2, and SEQ ID NO: 11 of HVR-H3.

[0171] The antibody may comprise a light chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 4, preferably while retaining HVR-L1 RASKSINKYLA (SEQ ID NO: 5), HVR-L2 SGSTLQS (SEQ ID NO: 6) and HVR-L3 QQHNEYPLT (SEQ ID NO: 7). The antibody may comprise a heavy chain variable domain amino acid sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 8, preferably while retaining HVR-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO: 10) and HVR-H3 ERDSTEVLP MDY (SEQ ID NO: 11).

[0172] Disclosed herein are methods of administering anti-Clq antibodies that inhibit the interaction between Clq and autoantibodies. In preferred embodiments, the anti-Clq antibodies promote clearance of Clq from circulation or tissues.

[0173] 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 promote the clearance of C1q from the circulation or tissues. In some embodiments, the anti-C1q antibodies inhibit the respective interactions at 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 C1q-C1s interactions, at approximately equimolar concentrations of C1q and anti-C1q antibodies. In other embodiments, the anti-C1q antibody is present in an amount of 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 1 In some embodiments, the anti-C1q antibody binds to C1q at a stoichiometry of 11:1, less than 10.5:1, less than 10:1, less than 9.5:1, less than 9:1, less than 8.5:1, less than 8:1, less than 7.5:1, less than 7:1, less than 6.5:1, less than 6:1, less than 5.5:1, less than 5:1, less than 4.5:1, less than 4:1, less than 3.5:1, less than 3:1, less than 2.5:1, less than 2.0:1, less than 1.5:1, or less than 1.0: 1. In some embodiments, the anti-C1q antibody binds to C1q at a stoichiometry of 20:1 to 1.0:1 or less than 1.0: 1. In some embodiments, the anti-C1q antibody binds to C1q at a stoichiometry of 6:1 to 1.0:1 or less than 1.0:1. In certain embodiments, the anti-C1q antibody binds to C1q with a binding stoichiometry of 2.5:1 to 1.0:1 or less than 1.0:1. In some embodiments, the anti-C1q antibody inhibits 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 C1q A chain. In other embodiments, the anti-C1q antibody binds to C1q B chain. In other embodiments, the anti-C1q antibody binds to C1q C chain. In some embodiments, the anti-C1q antibody binds to C1q A chain, C1q B chain, and / or C1q C chain.In some embodiments, the anti-C1q antibody binds to the globular domain of the C1q A chain, B chain, and / or C chain. In other embodiments, the anti-C1q antibody binds to the collagen-like domain of the C1q A chain, C1q B chain, and / or C1q C chain.

[0174] When an antibody of the present disclosure inhibits an interaction between two or more complement factors, such as an interaction between C1q and C1s or an interaction between C1q and C1r, the interaction that occurs in the presence of the antibody can 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% relative to a control in which the antibody of the present disclosure is not present. In some embodiments, the antibodies of the present disclosure reduce the 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% relative to a control in which the antibody of the present disclosure is not present.

[0175] In some embodiments, the antibodies of the present disclosure inhibit 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%, relative to a control in which the antibodies of the present disclosure are not present. Methods for measuring C2 or C4 cleavage are well known in the art. The EC values ​​of the antibodies of the present disclosure for C2 or C4 cleavage are as follows: 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, antibodies of the present disclosure inhibit C2 or C4 cleavage at approximately equimolar concentrations of CIq and corresponding anti-CIq antibodies.

[0176] In some embodiments, the antibodies of the present disclosure inhibit autoantibody-dependent 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 in which the antibodies of the present disclosure are not present. 50Values ​​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.

[0177] 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 in which the antibodies of the present disclosure are not present. Methods for measuring CDCC are well known in the art. EC values ​​for CDCC inhibition by the antibodies of the present disclosure are as follows: 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 present disclosure inhibit CDCC but not antibody-dependent cellular cytotoxicity (ADCC).

[0178] Humanized anti-complement C1q antibody

[0179] The humanized antibodies disclosed herein specifically bind to complement factor C1q and / or C1q protein in the C1 complex of the classical complement pathway. Humanized anti-C1q antibodies may specifically bind to human C1q, human and mouse C1q, rat C1q, or human C1q, mouse C1q and rat C1q.

[0180] All sequences mentioned in the following sixteen paragraphs are incorporated by reference into US Patent Application No. 14 / 933,517, the antibodies and related compositions disclosed therein are incorporated herein by reference.

[0181] In some embodiments, the human heavy chain constant region is a human IgG4 heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 47, or having at least 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% homology with SEQ ID NO: 47. According to Kabat numbering, the human IgG4 heavy chain constant region may comprise an Fc region having one or more modifications and / or amino acid substitutions. In this case, the Fc region comprises an amino acid substitution of leucine to glutamic acid at position 248, wherein such substitution inhibits the interaction of the Fc region with an Fc receptor. In some embodiments, the Fc region comprises an amino acid substitution of serine to proline at position 241, wherein such substitution prevents arm switching in the antibody.

[0182] Human IgG4(S241P L248E) The amino acid sequence of the heavy chain constant domain is: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:47).

[0183] 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.

[0184] The amino acid sequence of heavy chain variable domain variant 1 (VH1) is: The hypervariable region (HVR) of VH1 is in bold and underlined.

[0186] The amino acid sequence of heavy chain variable domain variant 2 (VH2) is: The hypervariable region (HVR) of VH2 is in bold and underlined.

[0188] The amino acid sequence of heavy chain variable domain variant 3 (VH3) is: The hypervariable region (HVR) of VH3 is in bold and underlined.

[0190] The amino acid sequence of heavy chain variable domain variant 4 (VH4) is: The hypervariable region (HVR) of VH4 is in bold and underlined.

[0192] The amino acid sequence of kappa light chain variable domain variant 1 (Vκ1) is: The hypervariable region (HVR) of Vκ1 is in bold and underlined.

[0194] The amino acid sequence of kappa light chain variable domain variant 2 (Vκ2) is: The hypervariable region (HVR) of Vκ2 is in bold and underlined.

[0195] The amino acid sequence of kappa light chain variable domain variant 3 (Vκ3) is: The hypervariable region (HVR) of Vκ3 is in bold and underlined.

[0197] The amino acid sequence of kappa light chain variable domain variant 4 (Vκ4) is: The hypervariable region (HVR) of Vκ4 is in bold and underlined.

[0199] 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 HVR-H1 GYHFTSYWMH (SEQ ID NO: 9), HVR-H2 VIHPNSGSINYNEKFES (SEQ ID NO: 10) and HVR-H3 ERDSTEVLPMDY (SEQ ID NO: 11).

[0200] 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.

[0201] The full-length antibody Mab2 comprises heavy chain variable domain variant 3 (VH3) (SEQ ID NO: 33) and kappa light chain variable domain variant 3 (Vκ3) (SEQ Id NO: 37). Mab2-Fab is the Fab of the Mab2 antibody.

[0202] In some embodiments, the humanized anti-C1q antibodies of the present disclosure include a heavy chain variable region containing a Fab region and a heavy chain constant region containing an Fc region, wherein the Fab region specifically binds to the C1q protein of the present disclosure, but the Fc region cannot bind to the C1q protein. In some embodiments, the Fc region is from a human IgG1, IgG2, IgG3 or IgG4 isotype. In some embodiments, the Fc region cannot induce complement activity and / or cannot induce antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the Fc region comprises one or more modifications, including but not limited to amino acid substitutions. In certain embodiments, the Fc region of the humanized anti-C1q antibodies of the present disclosure comprises an amino acid substitution at position 248 according to the Kabat numbering convention or a position corresponding to position 248 according to the Kabat numbering convention and / or at position 241 according to the Kabat numbering convention or a position corresponding to position 241 according to the Kabat numbering convention. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 inhibits the interaction of the Fc region with an Fc receptor. In some embodiments, the amino acid substitution at position 248 or a position corresponding to position 248 is an amino acid substitution of leucine to glutamic acid. In some embodiments, the amino acid substitution at position 241 or a position corresponding to position 241 prevents arm switching in the antibody. In some embodiments, the amino acid substitution at position 241 or a position corresponding to position 241 is an amino acid substitution of serine to proline. In certain embodiments, the Fc region of a humanized anti-C1q antibody of the present disclosure comprises the amino acid sequence of SEQ ID NO: 47, or an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homology to the amino acid sequence of SEQ ID NO: 47.

[0203] Anti-C1q Fab fragment (e.g., FabA)

[0204] Before the advent of recombinant DNA technology, proteolytic enzymes (proteases) that cleave polypeptide sequences were used to dissect 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 the antibody molecule into three fragments. Two of the fragments, called Fab fragments, are identical and contain antigen-binding activity. The Fab fragments correspond to the two identical arms of the antibody molecule, each consisting of a V fragment that is identical to the heavy chain. H and C HThe Fab fragments are composed of a complete light chain with 1 domain pairing. Other fragments do not contain antigen binding activity, but were initially observed to be easy to crystallize, and are therefore named Fc fragments (crystallizable fragments). When Fab molecules are compared with IgG molecules, it is found that Fab is superior to IgG for certain in vivo applications because it has higher mobility and tissue penetration ability, its circulation half-life is shortened, it can monovalently bind antigens without mediating antibody effector functions, and its immunogenicity is low.

[0205] Fab molecules are artificial fragments of approximately 50 kDa of Ig molecules, the heavy chain of which is separated by the constant domain C H 2 and C H 3 shortened. Two heterophilic (V L -V H and C L -C H 1) Domain interaction is the basis of the double-chain structure of Fab molecules, which is achieved through C L With C H Fab and IgG have the same antigen binding site formed by six complementary determining regions (CDRs), V L and V H Three each (LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3). CDRs define the highly variable antigen binding sites of antibodies. The highest sequence variation is found in LCDR3 and HCDR3, which are respectively controlled by V and V in the natural immune system. L and J L Gene or V H、 D H and J H Gene rearrangement. LCDR3 and HCDR3 usually form the core of the antigen binding site. The conserved region that connects and displays the six CDRs is called the framework region. In the three-dimensional structure of the variable domain, the framework region forms a sandwich of two antiparallel β-folds, which are connected by external hypervariable CDR loops and internal conserved disulfide bonds. The unique combination of stability and versatility of the antigen binding sites of Fab and IgG is the basis for their success in clinical practice for disease diagnosis, monitoring, prevention and treatment.

[0206] All anti-Clq antibody Fab fragment sequences are incorporated by reference into US Patent No. 15 / 360,549, the antibodies and related compositions disclosed therein are incorporated herein by reference.

[0207] In certain embodiments, the present disclosure provides binding sites comprising a heavy chain (V H / C H 1) and light chain (V L / C L), wherein the anti-C1q antibody Fab fragment has six complementary determining regions (CDRs), V L and V H Three each (HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3). The heavy chain of the antibody Fab fragment is truncated after the first heavy chain domain of IgG1 (SEQ ID NO: 39) and comprises the following amino acid sequence:

[0208]

[0209] The complementarity determining regions (CDRs) of SEQ ID NO: 39 are in bold and underlined.

[0210] The light chain domain of the antibody Fab fragment comprises the following amino acid sequence (SEQ ID NO: 40):

[0211]

[0212] The complementarity determining regions (CDRs) of SEQ ID NO: 40 are in bold and underlined.

[0213] 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.

[0214] Mab1-Fab is the Fab of the Mab1 (M1) antibody.

[0215] Mab2-Fab is the Fab of the Mab2 antibody.

[0216] Mab3-Fab is the Fab of the Mab3 antibody.

[0217] Nucleic acids, vectors and host cells

[0218] Antibodies suitable for use in the methods of the present disclosure can be produced using recombinant methods and compositions, for example, as described in U.S. Pat. No. 4,816,567. In some embodiments, isolated nucleic acids having nucleotide sequences encoding any of the antibodies of the present disclosure are provided. Such nucleic acids may encode a V L / C L The amino acid sequence of the anti-C1q antibody and / or V H / C H 1. In some embodiments, one or more vectors (e.g., expression vectors) containing such nucleic acids are provided. Host cells containing such nucleic acids can also be provided. The host cells can contain the following (e.g., have been transduced with the following): (1) a V vector encoding an antibody; L / C L The amino acid sequence and V containing antibody H / C H 1, or (2) a vector containing a nucleic acid encoding an antibody V L / C L A first vector containing a nucleic acid encoding an amino acid sequence of H / C H In some embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In some embodiments, the host cell is a bacterium, such as Escherichia coli.

[0219] Disclosed herein are methods for preparing anti-C1q antibodies. The methods include culturing a host cell of the disclosure containing a nucleic acid encoding an anti-C1q antibody under conditions suitable for antibody expression. In some embodiments, the antibody is subsequently recovered from the host cell (or host cell culture medium).

[0220] To recombinantly produce the humanized anti-C1q antibodies of the present disclosure, nucleic acids encoding the antibodies are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibodies).

[0221] Suitable vectors containing nucleic acid sequences encoding any antibody or fragment polypeptides thereof (including antibodies) disclosed herein include, but are not limited to, cloning vectors and expression vectors. Suitable cloning vectors can be constructed according to standard techniques, or can be selected from a large number of cloning vectors available in the art. Although the cloning vector selected can vary according to the host cell to be used, useful cloning vectors generally have the ability to self-replicate, can have a single target for a specific restriction endonuclease, and / or can carry genes for markers that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and derivatives thereof, mpl8, mpl9, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors can be obtained from commercial suppliers, such as BioRad, Stratagene, and Invitrogen.

[0222] Vectors containing nucleic acids of interest can be introduced into host cells by any of a number of suitable methods, including electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE dextran or other substances; microprojectile bombardment; lipid infection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus). The choice of the vector or polynucleotide to introduce will generally depend on the characteristics of the host cell. In some embodiments, the vector comprises a nucleic acid comprising one or more amino acid sequences encoding an anti-C1q antibody of the present disclosure.

[0223] Host cells suitable for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells. For example, the anti-C1q antibodies of the present disclosure can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. For expressing antibody fragments and polypeptides in bacteria (e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523; and Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describes the expression of antibody fragments in E. coli). In other embodiments, the antibodies of the present disclosure can be produced in eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NS0, Sp20 cells) (e.g., U.S. Patent Application No. 14 / 269,950; U.S. Patent No. 8,981,071; Eur J Biochem. 1991 Jan 1; 195(1):235-42). After expression, the antibodies can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0224] Pharmaceutical compositions and administration

[0225] The anti-C1q antibodies (eg, FabA) of the present disclosure can be administered in the form of a pharmaceutical composition.

[0226] Therapeutic formulations of the antibodies, antibody fragments and / or antibody derivatives of the present disclosure can be prepared for storage by mixing the antibodies having the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed.

[1980] ). Acceptable carriers, excipients or stabilizers are nontoxic 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., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residual yl) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; 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 ions; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®. TM 、PLURONICS TM or polyethylene glycol (PEG).

[0227] Lipofection or liposomes can also be used to deliver antibodies or antibody fragments or antibody derivatives into cells, where an epitope or minimal fragment of the binding domain that specifically binds to the target protein is preferred.

[0228] Antibodies can also be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization, 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).

[0229] The formulations for administration may be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0230] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers comprising antibodies, and the matrix is ​​in the form of formed articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., LUPRON DEPOT) (injectable microspheres composed of lactic acid-glycolic acid copolymers and leuprolide acetate) and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, the time for some hydrogels to release proteins is shorter.

[0231] The antibodies, antibody fragments and / or antibody derivatives and compositions of the present disclosure are typically administered by intravitreal administration.

[0232] The pharmaceutical composition may also contain (depending on the desired formulation) a pharmaceutically acceptable, non-toxic diluent carrier, which is defined as a vehicle commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, buffered water, normal 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 that approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, wetting agents, and detergents.

[0233] The composition can also include any of various stabilizers, such as antioxidants. When the pharmaceutical composition includes a polypeptide, the polypeptide can be compounded with various well-known compounds, which enhance the in vivo stability of the polypeptide, or enhance its pharmacological properties (e.g., increase the half-life of the polypeptide, reduce its toxicity, enhance other pharmacokinetic and / or pharmacodynamic characteristics, or enhance solubility or absorption). Examples of such modifications or compounding agents include sulfates, gluconates, citrates, and phosphates. The polypeptide of the composition can also be compounded with molecules that enhance its in vivo properties. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese) and lipids. Other guidance on formulations suitable for various types of administration can be found in Remington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa., 17th edition (1985). For a brief review of the method for drug delivery, see Langer, Science 249: 1527-1533 (1990).

[0234] Toxicity and therapeutic efficacy of active ingredients can be determined according to standard pharmaceutical procedures in cell cultures and / or experimental animals, including, for example, determination of LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Therapeutic agents that exhibit high therapeutic indices are preferred.

[0235] The data obtained from cell culture and / or animal studies and / or human clinical trials can be used to formulate a dosage range for humans. The dosage of the active ingredient is generally within a circulating concentration range that includes an ED50 with low toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized.

[0236] The components used to prepare the pharmaceutical composition 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, and more usually at least pharmaceutical grade). In addition, compositions intended for parenteral use are generally sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is generally substantially free of any potential toxic agents, particularly any endotoxins, which may be present during the synthesis or purification process. Compositions for parenteral administration are also generally substantially isotonic and are made under GMP conditions.

[0237] The compositions of the present disclosure can be administered using any medically appropriate procedure, for example, intravitreal injection.

[0238] Treatment

[0239] A role for complement in photoreceptor retinal diseases has been reported in multiple preclinical models of photoreceptor degeneration, where 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 drive photoreceptor degeneration are unclear. Based on the described role of C1q in microglia-mediated synaptic pruning in CNS development and disease, C1q could mark photoreceptor synapses in retinal degenerative conditions that lead to neuronal loss through aberrant microglia-mediated synapse elimination.

[0240] Using a photo-oxidative light injury model of photoreceptor degeneration, we provide evidence for C1q deposition at photoreceptor synapses and microglial phagocytosis of C1q-labeled synapses. We show a significant correlation between C1q levels in the retina and photoreceptor synapses and cell body loss. In addition, we show externalization of the C1q substrate phosphatidylserine (PS) at photoreceptor synapses, suggesting that PS may be involved in the recruitment of C1q at synapses. Finally, intravitreal treatment with an anti-C1q antibody reduced the levels of complement components in mildly injured retinas (confirming target engagement and neuroprotection following treatment).

[0241] Increased levels of the classical complement component C1q were demonstrated in retinal lysates from untreated and IgG Rd10 animals compared with WT ( Fig. 14B Anti-C1q treatment resulted in decreased C1q levels in retinal lysates compared with the IgG1-treated and untreated rd10 groups ( Fig. 14B ), demonstrating good measurable PK and C1q engagement in the retina. Photoreceptor synapses (a marker of BNS) were preserved after treatment with a C1q inhibitor ( Fig. 15B ).

[0242] 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, choroidal coloboma, fundus xanthophy, cone-rod dystrophy, and Leber congenital amaurosis) or retinal detachment in human patients.

[0243] Inherited retinal diseases (IRDs) are a group of conditions that can lead to severe vision loss or even blindness. Each IRD is caused by at least one gene that is not functioning properly. IRDs can affect individuals of all ages, can develop at different rates, and are rare. However, many are progressive, meaning that the symptoms of the disease get worse over time.

[0244] Many types of IRDs have been identified, and some have yet to be discovered. The most common types of IRDs include: retinitis pigmentosa / rod-cone dystrophy, choroidal coloboma, fundus xanthophy, cone-rod dystrophy, Leber congenital amaurosis, X-linked RP, and Usher syndrome. The common pathway is photoreceptor cell degeneration.

[0245] Retinitis pigmentosa (RP) / rod-cone dystrophy is a group of related eye diseases caused by variants in 60 genes that affect the retina. In people with RP, vision decreases as the retina's light-sensitive cells gradually die. Depending on the genes affected, the severity of the disease and how quickly it progresses can vary from person to person with RP. RP can first appear in childhood (early-onset RP) or in adulthood. The first sign of RP is often a loss of night vision, called night blindness. Later, RP causes blind spots in peripheral (side) vision. Over time, these blind spots develop into decreased peripheral vision. The disease progresses over time, eventually affecting central vision, also called tunnel vision, which is necessary for tasks such as reading, driving, and recognizing faces. Choroidal coloboma is a condition with progressive vision loss that primarily affects males. The first symptom of this condition is often night blindness, which can occur in early childhood. Over time, a person develops tunnel vision and loses the ability to see fine detail. These vision problems are due to the loss of cells in the retina and the nearby network of blood vessels, called the choroid. The visual impairment of choroidal coloboma worsens over time, but the rate of worsening varies among affected individuals. The condition may lead to complete blindness by late adulthood.

[0246] Fundus xanthosphagia is also known as Stargardt's macular dystrophy. This disease causes damage to the macula, a small area in the center of the retina that is responsible for sharp, straight-ahead vision. The disease usually causes central vision loss during childhood or adolescence. Sometimes, vision loss may not be noticed until adulthood. Rarely, people with this disease will experience complete loss of vision.

[0247] Cone-rod dystrophy (CRD) is a group of more than 30 IRDs that affect cones and rods. Cones and rods are light-sensing cells in the retina. People with this condition lose vision over time as the cones and rods progressively degenerate. The first symptoms usually occur in childhood and may include blurred vision and intense sensitivity to light (called photophobia). These symptoms are followed by blind spots in the center of vision, loss of the ability to see color, and loss of side or peripheral vision. Most people with this condition lose a significant amount of vision by mid-adulthood.

[0248] Leber congenital amaurosis (LCA) is an eye disease that primarily affects the retina. The retina is a layer of the eye that acts like film in a camera, capturing visual images and sending electrical signals to the brain. LCA is one of the earliest forms of IRD. People with this condition usually have severe visual impairment in infancy. LCA is also associated with other vision problems, such as photophobia: increased sensitivity to light; nystagmus: uncontrollable movement of the eyes; extreme farsightedness: the inability to see close objects, such as a book or a watch face; unresponsive pupils: the pupils of people with LCA do not react normally to light. Instead, the pupils open and close more slowly than normal, or they may not react to light at all; corneal malformations: the cornea of ​​people with LCA may be cone-shaped and abnormally thin; and strabismus: the muscles of the eye do not form or work properly, causing the eyes to look at two different places at the same time.

[0249] 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 may also be clinically affected, although the phenotype is usually much more severe than in males. The phenotypic differences in female carriers are attributed to a pattern of random inactivation of the X chromosome carrying the wild-type gene during retinal tissue development, which is modulated by other genetic and environmental factors. XLRP is most commonly caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene on the X chromosome. It is characterized by early onset and rapid progression of visual loss, leading to legal blindness by the end of the third decade. Less common forms of XLRP are caused by mutations in the RP2 gene and the OFD1 gene. Mutations in the RPGR gene may be associated with a rod-cone or cone-rod dystrophy phenotype. Most cases present with a rod-cone dystrophy-type disease progression, in which central visual acuity is initially less impaired than peripheral visual field loss. However, during the early stages of the disease, some patients also present with early cone involvement and corresponding impairment of central visual acuity. During advanced stages of the disease, the fovea is eventually affected by subsequent degeneration of cone photoreceptor cells.

[0250] Usher syndrome (also known as Hallgren syndrome, Arthur-Hallgren syndrome, retinitis pigmentosa-dyshearing syndrome or malnutrition retinal hearing syndrome) is a rare hereditary disease caused by mutations in any of at least 11 genes, resulting in a combination of hearing loss and visual impairment. This is the main cause of deaf-blindness and is currently incurable. It can cause deafness or hearing loss and an eye disease called retinitis pigmentosa (RP). Sometimes, it can also cause balance problems. According to the genes and morbidity that cause deafness, Usher syndrome is divided into three subtypes (I, II and III). All three subtypes are caused by genetic mutations related to inner ear and retinal function. These mutations are inherited in an autosomal recessive manner.

[0251] Retinal detachment describes a condition in which a thin layer of nerve tissue at the back of the eye (the retina) detaches 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. Now that the retina is separated from the RPE, the photoreceptor cells degenerate, leading to vision loss. If the central retina detaches, and the longer the retinal detachment goes untreated, the greater the risk of permanent blindness in the affected eye. Warning signs of retinal detachment may include one or all of the following: the sudden appearance of floaters and flashes of light and decreased vision. Retinal detachment occurs in approximately 10-12 cases per 100,000 people each year. In approximately 50% of cases, the central retina detaches. When the central retina detaches, recovery of vision only reaches approximately 50% of pre-detachment vision, despite successful reattachment of the retina. The reason for this limited vision recovery is photoreceptor cell degeneration.

[0252] Genetic or pharmacological inhibition of the classical complement pathway results in increased photoreceptor survival and improved retinal function. However, the mechanisms by which C1q and the classical complement pathway drive photoreceptor degeneration remain unclear. Based on the role of C1q in microglia-mediated synaptic pruning in CNS development and disease, we hypothesized three mechanisms of photoreceptor damage mediated by C1q: (1) C1q marks photoreceptor synapses in retinal degenerative diseases, leading to neuronal loss through aberrant microglia-mediated synapse elimination; (2) C1q is activated by waste products of damaged photoreceptors (phosphatidylserine, C-reactive protein, and altered photoreceptor cell membranes), leading to impaired recruitment of phagocytes, which also recruit additional C1q, and (3) activation of the entire classical complement pathway and formation of the membrane attack complex (MAC) leading to cell lysis.

[0253] C1q recognizes certain pathogens, modifications of autoantigens, antigen-bound antibodies or specific molecules on the cell surface. During normal aging, C1q accumulates at synapses (possibly those weakened by age or neuronal stress) and subsequent various pathophysiological stimuli can trigger activation of the classical complement cascade, leading to inappropriate elimination of synapses. This abnormal inflammatory response associated with synaptic removal is known as complement-mediated neurodegeneration (CMND). CMND is associated with Alzheimer's disease, schizophrenia, Huntington's disease, frontotemporal dementia, spinal muscular atrophy and glaucoma. With retinal degenerative stress, C1q activation leads to synaptic elimination and causes loss of RGCs and optic nerves.

[0254] Using a photooxidative injury model of photoreceptor degeneration, we provide evidence for C1q deposition at photoreceptor synapses and demonstrate microglial engulfment of C1q-labeled synapses. We show a significant correlation between C1q levels and cell body loss in the retina and at photoreceptor synapses. We also show externalization of the C1q substrate phosphatidylserine (PS) at photoreceptor synapses, suggesting that PS may be involved in the recruitment of C1q at synapses. Finally, we show that intravitreal treatment with an anti-C1q antibody reduces the levels of complement components in light-damaged retinas. Furthermore, we demonstrate C1q deposition at photoreceptor synapses in human geographic atrophic retinal tissue, suggesting that this mechanism is relevant to humans.

[0255] 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, choroidal coloboma, fundus xanthophy, cone-rod dystrophy, and Leber congenital amaurosis) or retinal detachment in human patients.

[0256] Such methods comprise administering to a patient by 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). Such methods further comprise administering to the patient by 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 an amino acid sequence of SEQ ID NO:5, HVR-L2 having amino acids of SEQ ID NO:6, and HVR-L3 having amino acids of SEQ ID NO:7; and a heavy chain variable domain comprising HVR-H1 having an amino acid sequence of SEQ ID NO:9, HVR-H2 having amino acids of SEQ ID NO:10, and HVR-H3 having amino acids of SEQ ID NO:11. NO:11. The composition administered may include about 1 mg to about 5 mg of the anti-C1q antibody. The composition administered may include 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 the anti-C1q antibody. The composition administered may include about 5 mg of the anti-C1q antibody. The composition administered may include about 10 mg of the 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, and wherein the light chain variable domain comprises HVR-L1 having an amino acid sequence of SEQ ID NO:5, HVR-L2 having amino acids of SEQ ID NO:6, and HVR-L3 having amino acids of SEQ ID NO:7. 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, and wherein the heavy chain variable domain comprises HVR-H1 having an amino acid sequence of SEQ ID NO: 9, HVR-H2 having amino acids of SEQ ID NO: 10, and HVR-H3 having amino acids of SEQ ID NO: 11. 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 and a heavy chain variable domain, the light chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs:4 and 35-38, and wherein the light chain variable domain comprises HVR-L1 having an amino acid sequence of SEQ ID NO:5, HVR-L2 having amino acids of SEQ ID NO:6, and HVR-L3 having amino acids of SEQ ID NO:7, the heavy chain variable domain comprises an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs:8 and 31-34, and wherein the heavy chain variable domain comprises HVR-H1 having an amino acid sequence of SEQ ID NO:9, HVR-H2 having amino acids of SEQ ID NO:10, and HVR-H3 having amino acids of SEQ ID NO:11. In some embodiments, the antibody comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprises an amino acid sequence selected from SEQ ID NO: 4 and 35-38, and the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NO: 8 and 31-34. In some embodiments, the antibody can be a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, an antibody fragment or an antibody derivative thereof. The antibody fragment can be a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a 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.

[0257] In some embodiments, the antibody is administered once a week, once every other week, once every three weeks, once a month, once every 4 weeks, once every six weeks, once every 8 weeks, once every other month, once every 10 weeks, once every 12 weeks, once every three months, or once every 4 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.

[0258] In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 2.5 mg / eye once a month, once every 4 weeks, once every 6 weeks, or once every other month.

[0259] In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 5 mg / eye once a month, once every 4 weeks, once every 6 weeks, or once every other month. In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 5 mg / eye once a month or once every 4 weeks. In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 5 mg / eye once every 6 weeks. In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 5 mg / eye once every other month or once every 8 weeks.

[0260] In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 10 mg / eye once a month, once every 4 weeks, once every 6 weeks, or once every other month. In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 10 mg / eye once a month or once every 4 weeks. In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 10 mg / eye once every 6 weeks. In certain preferred embodiments, FabA is administered as an IVT injection at a dose of 10 mg / eye once every other month or once every 8 weeks.

[0261] FabA injections were performed by trained and experienced physicians using aseptic technique via IVT injection.

[0262] The anti-C1q antibody may inhibit the interaction between C1q and autoantibodies, between C1q and C1r, or between C1q and C1s, or may promote the clearance of C1q from the circulation or tissues. In some embodiments, the dissociation constant (K) of the anti-C1q antibody is D) is from 100 nM to 0.005 nM or less. In some embodiments, the anti-C1q antibody binds to C1q with a binding stoichiometry of from 20:1 to 1.0:1 or less, a binding stoichiometry of from 6:1 to 1.0:1 or less, or a binding stoichiometry of from 2.5:1 to 1.0:1 or less.

[0263] The method inhibits the biological activity of C1q, for example, (1) the binding of C1q to autoantibodies, (2) the binding of C1q to C1r, (3) the binding of C1q to C1s, (4) the binding of C1q to phosphatidylserine, (5) the binding of C1q to pentraxin-3, (6) the binding of C1q to C-reactive protein (CRP), (7) the binding of C1q to globular C1q receptor (gC1qR), (8) the binding of C1q to complement receptor 1 (CR1), (9) the binding of C1q to β-amyloid protein, or (10) the binding of C1q to calreticulin. In other embodiments, the biological activity of C1q is (1) activation of the classical complement activation pathway, (2) reduction in lysis and / or reduction in C3 deposition, (3) activation of antibodies and complement dependent cytotoxicity, (4) CH50 hemolysis, (5) reduction in erythrocyte lysis, (6) reduction in erythrocyte phagocytosis, (7) reduction in dendritic cell infiltration, (8) inhibition of complement-mediated erythrocyte lysis, (9) reduction in lymphocyte infiltration, (10) reduction in macrophage infiltration, (11) reduction in antibody deposition, (12) Reduction in neutrophil infiltration, (13) Reduction in platelet phagocytosis, (14) Reduction in platelet lysis, (15) Improvement in transplant graft survival, (16) Reduction in macrophage-mediated phagocytosis, (17) Reduction in autoantibody-mediated complement activation, (18) Reduction in red blood cell destruction caused by transfusion reactions, (19) Reduction in red blood cell lysis caused by alloantibodies, (20) Reduction in hemolysis caused by transfusion reactions, (21) Reduction in alloantibody-mediated platelet lysis, (22) Improvement in anemia , (23) reduction in eosinophilia, (24) reduction in C3 deposition on red blood cells (e.g., reduction in deposition of C3b, iC3b, etc. on RBCs), (25) reduction in C3 deposition on platelets (e.g., reduction in deposition of C3b, iC3b, etc. on platelets), (26) reduction in anaphylatoxin production, (27) reduction in autoantibody-mediated blister formation, (28) reduction in autoantibody-induced erythema, (29) reduction in red blood cell destruction caused by transfusion reactions, (30) reduction in blood transfusion reactions caused by transfusion reactions reduction in platelet lysis, (31) reduction in mast cell activation, (32) reduction in mast cell histamine release, (33) reduction in vascular permeability, (34) reduction in complement deposition on the transplant graft endothelium, (35) B cell antibody production, (36) dendritic cell maturation, (37) T cell proliferation, (38) cytokine production, (39) microglial activation, (40) Arthritis reaction, (41) reduction in anaphylatoxin production in the transplant graft endothelium, or (42) activation of complement receptor 3 (CR3 / C3) expressing cells.

[0264] In some embodiments, CH50 hemolysis includes 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.

[0265] 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.

[0266] It is expected that the composition for in vivo use can be obtained and used under the guidance of a physician. The dosage of the therapeutic formulation can vary widely depending on the nature of the disease, the frequency of administration, the mode of administration, the clearance of the agent from the host body, and the like.

[0267] As used herein, "chronic administration," "chronic treatment," "chronically treating," or similar grammatical variations thereof, refers to a treatment regimen that is used to maintain a certain threshold concentration of the therapeutic agent in the patient's eye so as to completely or substantially inhibit the patient's systemic complement activity for an extended period of time. Thus, a patient treated chronically with an anti-C1q antibody can be treated for a period of time greater than or equal to 2 weeks (e.g., 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, , 48 weeks, 49 weeks, 50 weeks, 51 weeks or 52 weeks; 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months; or 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, 5 years, 5.5 years, 6 years, 6.5 years, 7 years, 7.5 years, 8 years, 8.5 years, 9 years, 9.5 years, 10 years, 10.5 years or 12 years or the rest of the patient's life), wherein the amount of the antibody and the frequency of administration are sufficient to maintain an antibody concentration in the eye of the patient that inhibits or substantially inhibits complement activity throughout the patient's body. In some embodiments, the antibody can be administered to a patient in need for a long term in an amount and frequency effective to maintain serum hemolytic activity less than or equal to 20% (e.g., 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6% or even less than 5%). In some embodiments, the antibody can 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 of LDH (e.g., 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6% or even less than 5%).

[0268] Therapeutic agents, eg, anti-Clq antibodies, can be incorporated into a variety of formulations for therapeutic administration by combining with appropriate pharmaceutically acceptable carriers or diluents.

[0269] Example

[0270] Example 1: Method

[0271] Photooxidation White Light Damage Model

[0272] Male Balb / C mice (12 weeks old; Charles River Laboratories) were used for the studies described in Examples 2-7. Animals were dark-adapted overnight in the experiment, and their dark period was extended by 3-4 hours at a time until the start of light induction (acute: 25k lux, 4 hours; mild: 5k Lux, 30 minutes). Acute light settings were used for model characterization (results shown in Figure 1). All other experiments used mild light settings (results shown in Figures 2-5). Animals were sacrificed and tissues were collected at baseline, day 1, day 3, and day 7.

[0273] Rd10 mouse model

[0274] Rd10 mice were housed in darkness from birth and transferred to a house with a light-controlled environment (~200 lux of normal daytime light cycles) at P30-P31.

[0275] Anti-C1q antibody treatment (or IgG control antibody) was administered intraperitoneally (ip) at a dose of 100 mg / kg twice a week starting from P12. For ip injections, mice were lightly restrained by hand and the abdomen was punctured with an 8 mm, 31 gauge needle to deliver the antibody. Animals were sacrificed at P30, P33, P38 and tissues were collected.

[0276] Terminal plasma and ocular tissue collection

[0277] Animals were anesthetized and about 300-500 μl of terminal whole blood was collected by cardiac puncture. The blood was immediately placed in a K2EDTA test tube and centrifuged at 5,000 x g for 10 minutes to obtain plasma. The plasma was transferred to a 1.7 mL spring-top test tube and stored at -80 ° C until shipped to the customer on dry ice. For biochemical evaluation, animals were perfused through the heart with saline and eyes were removed. Retinas / RPE were separated, weighed, quickly frozen and stored at -80 ° C. For histopathological evaluation, animals were perfused through the heart with saline and then with 4% PFA (minimum 10 minutes / minimum 50 milliliters). After saline / PFA perfusion, eyes were collected. The posterior eye cup was dropped in 4% PFA and fixed for 2 hours, transferred by a series of sucrose gradients (10%, 20%, 30%), embedded in optimal cutting temperature compound (OCT) and frozen.

[0278] Intravitreal injection

[0279] Animals were anesthetized with isoflurane. Eyes were injected with a 32G needle inserted into the mid-vitreous under a surgical microscope. For PSVue labeling, 2 μl of 1 mM PSVue was injected into the eye 6 hours before tissue collection. The contralateral eye was used as an uninjected 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 one day before light exposure, and tissue collection was performed at baseline, day 3, and day 5.

[0280] ELISA

[0281] Animals were perfused transcardially with saline and eyes were removed. Retinas / RPE were isolated, weighed, snap frozen and stored at -80°C. Frozen tissues were homogenized using a pestle motor in lysis buffer (25mM Hepes, 0.1M NaCl, 1% Triton X100, complete protease inhibitor cocktail; or 10mM EDTA and Thermo Scientific #A32965 in Tris-buffered saline). Proteins were quantified using a Micro BCA assay kit (Thermo Scientific #23235). Complement signals were determined using a standard sandwich ELISA assay. In brief, black 96-well ELISA plates (Costar #3925) were coated with 75 μL of bicarbonate buffer (pH 9.4) containing 10ug / mL capture antibody overnight at 4°C. 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 plate (75 μL / well) after removing the blocking solution. The plate / plate was shaken at 300 rpm at 4°C overnight. The next day, the plate was washed three times with wash buffer (dPBS containing 0.05% Tween) and incubated with detection antibody (75 μl / well) at room temperature for 1 hour. After incubation, the plate was washed three times with wash buffer, developed by adding alkaline phosphatase substrate (Life Technologies, T2214), and read using a photometer. For PK determination, mouse C1q (M099, Complement Tech) and AP-conjugated anti-mouse antibody (115-055-071, Jackson Immuno Research) were used as capture and detection antibodies, respectively. For C1q determination, in-house capture antibody (JL1 clone, Atum) and detection antibody (M1-AP, antibody solution 1:3000) were used. C1s determination was performed using anti-mouse C1s (LSBio, 2ug / ml) and anti-mouse C1s-AP (LSBio, 1:5000) as capture and detection antibodies, respectively. C3d determination 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 determination was performed using a kit (Abcam, ab108792). Standards were fitted using 4PL logistic fitting, and unknowns were converted to concentrations, dilution correction was performed, and then graphed using GraphPad Prism.

[0282] Immunohistochemistry

[0283] Animals were transcardially perfused with saline followed by 4% PFA (minimum 10 min / minimum 50 ml). After saline / PFA perfusion, eyes were collected. The posterior eye cup was fixed in 4% PFA for 2 h, transferred through a series of sucrose gradients (10%, 20%, 30%), embedded in optimal cutting temperature compound (OCT) and frozen. After embedding, 10 μm thick sections were cut using a cryostat, serially collected onto 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 at 4°C overnight. The next day, sections were washed, incubated with appropriate Alexa-fluorophore-conjugated secondary antibodies (ThermoFisher Scientific) for 2 h, washed and coverslipped with Fluoromount G (Southern Biotech). All washing steps were performed in PBS for 3 × 10 minutes. The nucleus was 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 using an epifluorescence microscope (Leica) at 10x or 20x magnification and a confocal microscope (Leica, Stellaris 5) at 63x magnification. Images were processed using ImageJ.

[0284] C1q synaptic colocalization and phagocytosis analysis

[0285] Two Z-stack images were taken for each slice (Z-stack size: 0.7 um, 12 planes), and at least three slices from each animal were analyzed. A 3D volume surface rendering of each z-stack was created (Imaris Software). The retinal OPL was selected as the region of interest. The surface rendered images were used to identify the surface and volume of microglia, as well as synapses and C1q elements. C1q-labeled synapses were quantified based on proximity (C1q and bassoon element distance ≤ 400 μm). Engulfed synapses were quantified based on overlap (bassoon-Iba1 overlap ≥ 70%) and calculated as the number of engulfed C1q-labeled synapses / total number of synapses.

[0286] Phosphatidylserine (PS)-C1q binding assay

[0287] Phosphatidylserine (PS) and phosphatidylcholine (PC) lipid microparticles were purchased from Echelon Biosciences (#P-B1PS and P-B1PC). Purified human C1q was purchased from Complement Technology (#A099). Ninety-six (96) fully transparent round-bottom plates were used for the assay (VWR#353227). Microparticle suspensions were prepared according to the manufacturer's instructions. Briefly, the microparticles were first thoroughly vortexed to ensure uniform bead suspension. Washing, dilution, titration, and incubation steps were performed in Annexin V binding buffer (Thermo Fisher #BMS500BB). All washing steps were performed at a centrifugation speed of 10,000 g for 10 minutes. The beads were diluted 20 times before use. For each test point, 150 μL of bead suspension (approximately 100,000 beads) was used. The microparticles were washed twice and resuspended in binding buffer. Fifty microliters per well was considered the reaction volume. C1q titrations were prepared in binding buffer (1:10 dilution, 100-0.1 μg / mL). 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 1 volume of running buffer (PBS, 1% BSA, 2mM EDTA). The supernatant was decanted and the microparticles were resuspended in running buffer (100 ul / 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 running buffer (150 μl) and analyzed by flow cytometry using the green channel (FITC) for microparticles and the far red channel (APC) for C1q binding.

[0288] Complement deposition assay

[0289] Washing, dilution, titration and incubation steps were performed in GVB++ buffer (Complement Technology #B100). PS and PC lipid microparticles were washed twice and resuspended in an appropriate volume, considering approximately 100,000 beads per test point. Human serum titrations were prepared in GVB++. Ninety-six (96) fully transparent round bottom plates were used for the assay (VWR 353227). GVBEDTA (Complement Technologies #B105) was used as a negative control buffer. For a final volume of 33.3 μl, the reaction mixture included a 1:1:1 ratio of buffer (GVB++ or GVB EDTA, + / - titrated anti-C1q antibody): lipid microparticle: human serum. After incubation at 37°C for 30 minutes, the plates were washed twice with GVB++ (150 μl). The microparticles were resuspended in running 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 minutes at 4° C. After incubation, the microparticles were washed twice, resuspended in running buffer (150 μl) and analyzed by flow cytometry using the green channel (FITC) for microparticles and the far red channel (APC) for C1q or C4 deposition.

[0290] Human GA donor tissue acquisition and immunohistochemistry

[0291] Human donor eyes were obtained from the San Diego Eye Bank in California, USA within 24 hours after death. Clinical records and family questionnaires were conducted on all donors. Human eyes were fixed in 4% for 2 hours and then transferred to PBS overnight. The next day, the posterior eye cup was cryoprotected in 30% sucrose for 24-48 hours. The macula was then separated using a 6mm diameter anatomical trephine (Biomedical Research Instruments, MD, USA). The same trephine was used to sample the temporal, nasal, superior and inferior regions. Retinal samples were embedded in OCT and frozen. After embedding, 10 μm thick sections were cut using a cryostat, continuously collected 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 Systems, 1:500). Rabbit IgG isotype antibody (ThermoFisher) was used as a negative control for C1q staining. Images were captured using an epifluorescence microscope (Leica) at 10x magnification and a confocal microscope (Leica, Stellaris 5) at 63x magnification. Images were processed using ImageJ.

[0292] Statistical analysis

[0293] All data are presented as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism using unpaired Student's t-test or one-way analysis of variance (ANOVA) or two-way ANOVA followed by Bonferroni or Dunnet or Sidak or Tukey's post hoc test. Data are presented as mean ± SD. All p values ​​are shown in the figure legends and were considered statistically significant when less than 0.05.

[0294] Example 2: Photoreceptor cell synaptic loss and microglial increase in the photooxidative light damage model

[0295] The presynaptic marker Bassoon was used to identify synapses and synaptic density was measured using epifluorescence. The number of rows of photoreceptor nuclei was used as a measure of photoreceptor survival. Iba1 (calcium-binding protein specifically expressed in microglia) was used as a pan-microglia / macrophage marker. CD68 (lysosomal protein) was used to identify reactive phagocytic microglia. After photooxidative damage, progressive photoreceptor synapse and cell body loss was observed ( Figures 1A-1C ), and increased microglial reactivity ( Figures 1D-1FNotably, the distribution of phagocytic microglia in the synaptic layer peaked on day 1, the time point at which significant synaptic loss was first observed ( Figure 1E ).

[0296] Example 3: Increased C1q levels at photoreceptor synapses in a photooxidative light damage model of photoreceptor cell degeneration Photoreceptor synaptic loss

[0297] Increased levels of the initial classical complement components C1q and C1s and the downstream activation product C3d were observed in retinal lysates from light-exposed animals by standard ELISA ( Figure 2A-2C ). Next, the distribution of C1q in the retina was investigated by immunofluorescence. Figure 2D As shown in Figure 2, C1q (i) colocalized with Iba1 (iii) and Bassoon (iv), confirming the expression of C1q in microglia / macrophages and its expression / deposition at synapses. A significant negative correlation was observed between C1q expression and photoreceptor synaptic density (Figure 2G, Pearson correlation coefficient = -0.72, p = 0.00003), suggesting a causal relationship ( Figure 2E ).

[0298] Example 4: Microglial phagocytosis of C1q-labeled presynaptic elements in photoreceptor cells after photooxidative damage

[0299] To assess microglial phagocytosis of C1q-labeled synapses, we triple-labeled retinal sections from intact and light-exposed mice for C1q, Bassoon, and Iba1 ( Figure 3 A). High-resolution imaging using confocal microscopy. Phagocytosis analysis using 3D reconstruction and surface rendering software ( Figure 3 B). A significant decrease in synaptic density ( Figure 3 C) A significant increase in the percentage of synapses labeled with C1q ( Figure 3 D) and a significant increase in microglia engulfing C1q-labeled synapses in mildly injured retinas compared with uninjured retinas ( Figure 3 E).

[0300] Example 5: Phosphatidylserine binds to C1q and is externalized at photoreceptor cell synapses after photooxidative damage

[0301] 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 injury model (Scott-Hewitt N et al., The EMBO Journal, 2020). Retinal tissue was processed and analyzed using IHC on day 3 after light exposure. Increased PSVue labeling was detected in the OPL and ONL of mildly injured retinas compared to uninjured ones ( Figure 4A). High-resolution confocal imaging and 3D reconstruction / surface rendering confirmed colocalization of PSVue labeling with C1q and Bassoon ( Figure 4 A). Notably, the PSVue-positive surface appeared near the bassoon and C1q-positive surfaces in a sandwich-like manner ( Figure 4 A iii), demonstrating PS externalization and C1q interaction at synapses.

[0302] To evaluate whether PS directly binds to C1q and activates the complement cascade, a C1q binding and complement deposition assay was performed using PS-lipid-green-fluorescent-microparticles. Phosphatidylcholine (PC)-lipid green fluorescent microparticles were used as negative controls. After incubation with C1q at a concentration of 10 ng / mL, direct binding of C1q to PS lipid microparticles but not PC was observed ( Figure 4 B). Deposition of C1q and C4 was observed on PS lipid particles, but not on PC lipid particles. Almost no deposition was observed in the negative control group ( Figure 4 C and Figure 4 D). Titration of anti-C1q neutralizing antibodies (Mab1-Fab) in serum resulted in a decrease in C1q and C4 deposition on PS lipid particles ( Figure 4 E and Figure 4 F), showing that competitive binding of anti-C1q antibody reduces PS-mediated complement activation.

[0303] Example 6: Anti-C1q intravitreal treatment reduces retinal complement component levels after photooxidative light damage

[0304] Intravitreal administration of a neutralizing optimized mouse anti-C1q antibody (Mab3, optimized from Mab1) was performed one day prior to exposure to light damage. Detectable drug levels and selected complement component levels were assessed by standard ELISA on day 3 after light damage. Measurable drug levels were found in retinal lysates of animals treated with anti-C1q, but not in IgG1-treated or untreated groups ( Figure 5 A). Levels of classical complement components C1q and C1s and the downstream activation component C3d were elevated in retinal lysates from light-exposed animals in both untreated and IgG-treated groups compared with untreated controls ( Figure 5 B-5D). Significant decreases in C1q, C1s, and C3d were observed after anti-C1q treatment compared with IgG antibodies ( Figure 5 B-5D). Validation studies confirmed target engagement and assessed neuroprotection after treatment.

[0305] Example 7: Expression and deposition of C1q at photoreceptor synapses in human GA donor retinas

[0306] GA donor eyes were obtained and histological assessment of C1q distribution across retinal layers was performed. Synaptic integrity was assessed by immunofluorescence in the macular region of GA donor versus healthy donor retinas. Reduced immunoreactivity for the presynaptic marker Vglut1 and increased labeling of C1q in the photoreceptor synaptic layer compared with healthy donors confirmed synaptic loss and C1q accumulation in GA retinas ( Figure 6 Finally, triple immunolabeling of C1q (grey), the presynaptic marker Vglut1, and the postsynaptic marker Homer1 confirmed the colocalization of C1q with photoreceptor synapses in human GA donor retinas ( Figure 6 C).

[0307] Example 8: Evaluation of FabA in nonclinical studies

[0308] The FabA drug product is a sterile isotonic liquid for IVT injection.

[0309] FabA is supplied in sterile single-use vials for IVT injection.

[0310] FabA has been subjected to an extensive series of in vitro and in vivo pharmacological studies.

[0311] Antibodies Mab1, Mab1-Fab, and Mab2 were active in a mouse model of acute glaucoma and prevented retinal ganglion cell and / or nerve fiber loss. In a mouse model of photooxidative light-induced damage, intravitreally administered Mab1 prevented photoreceptor cell loss and retinal functional connectivity in the eye.

[0312] FabA GLP studies included a single-dose rat ocular toxicology study and three repeat-dose cynomolgus monkey ocular toxicology studies. The route of administration for the toxicology studies was IVT injection. In the single-dose and two-dose (once-monthly) IVT GLP studies, FabA did not show any evidence of adverse ocular toxicity, with a no observed adverse effect level (NOAEL) of 5 mg / eye (equivalent to a 10 mg human dose) in the monthly study in cynomolgus monkeys and 0.05 mg / eye (equivalent to a 10 mg human dose) in the single-dose rat study. In the 26-week chronic ocular toxicology study in cynomolgus monkeys, adverse ocular changes were related to the dual injection procedure and / or were determined to be anti-drug antibody (ADA)-mediated and not a direct effect of FabA IVT administration.

[0313] Pharmacokinetic evaluation of FabA was performed in the serum and vitreous of rats and cynomolgus monkeys. C1q levels were measured in the vitreous as a PD marker for FabA inhibition of C1q in cynomolgus monkeys. PK / PD and TK / PD studies in cynomolgus monkeys demonstrated robust ocular PD effects consistent with FabA drug exposure levels in the vitreous.

[0314] Binding and affinity of FabA and precursor molecules to human C1q

[0315] The C1q binding affinity of FabA and precursor molecules was also examined by ELISA. All molecules (Mab2-Fab, FabA and Mab2, Mab1 and Mab1-Fab) showed affinity for human C1q with half maximal effect concentrations (EC50) ranging from 2.2-4.9 ng / mL (20-95 pM). The EC50 for FabA binding to C1q was 2.5 ng / mL.

[0316] Effects on IgM-mediated hemolysis of erythrocytes

[0317] The activity of FabA, Mab2, and Mab2-Fab in functionally inhibiting classical complement-dependent hemolysis of IgM-opsonized RBCs in human serum was measured ( Fig. 9 ). The three molecules exhibited nearly identical potency, consistent with their equivalent binding affinities. The half-maximal inhibitory concentration (IC50) for FabA inhibition of IgM-coated RBC hemolysis was 0.62 μg / mL (approximately 12 nM).

[0318] In vivo pharmacology studies

[0319] Anti-C1q antibody treatment prevents optic nerve damage in an acute glaucoma mouse model

[0320] In mice, injection of polystyrene beads into the anterior chamber of the eye resulted in a dramatic increase in IOP, loss of retinal ganglion cells, and optic nerve damage within 2 weeks. Mab1, Mab1-Fab, and Mab2 were injected intravitreally into mice one day before and 7 days after the IOP increase. 2 μL of 10 mg / mL antibody or saline was administered at each time point. The antibody concentration was 2000-4000 μg / mL based on a vitreous volume of 5-10 μL in the mouse eye. Optic nerves were collected 2 weeks after injury, and the number of intact and damaged axons was quantified. In this induced glaucoma mouse model, anti-C1q antibody treatment resulted in protection against RGC loss and / or retinal nerve fiber damage ( Fig.10 ).

[0321] Anti-C1q antibody treatment prevents photoreceptor cell damage in a photooxidative light-induced damage model

[0322] When mice were exposed to 100Klux of natural white light LEDs for 1-7 days, photooxidative damage led to retinal photoreceptor loss. In this model, C1qa gene expression increased in a time-dependent manner within 3-7 days, which was associated with photoreceptor cell death and microglia / macrophage recruitment. C1qa- / - mice showed less photoreceptor cell death, reduced recruitment of microglia / macrophages to photoreceptor lesions, and higher visual function 14 days after light damage induction, but not on day 7. IVT administration of Mab1 antibody on day 7 after light damage reduced photoreceptor cell loss and maintained retinal function, as measured by electroretinogram (Figure 11). Mice were administered 1μL of 7.5mg / mL antibody, which is equivalent to a concentration of 750-1500μg / mL in the vitreous. In contrast, systemic delivery of Mab1 at 100mg / kg on days 0, 4, and 8 had no effect on photoreceptor cell loss or function. Retinal C1q is predominantly expressed by subretinal microglia / macrophages localized to the outer retina in early AMD and mouse retina. Thus, protection by anti-C1q antibodies suggests that C1q has a clear role in the initiation of photoreceptor cell damage and in the classical complement cascade in the pathogenesis of the human disease GA.

[0323] Safety Pharmacology

[0324] In a 26-week study in cynomolgus monkeys, systemic exposure after chronic IVT dosing did not exceed 86.3 ng / mL, while in a 26-week study in cynomolgus monkeys, systemic exposure of Mab2 with the same CDRs exceeded 1 mg / mL after once-weekly IV dosing at a NOAEL of 200 mg / kg.

[0325] Thus, safety pharmacology endpoints of the full-length antibody Mab2 were demonstrated in a 4-week repeat-dose GLP toxicity study in cynomolgus monkeys up to 200 mg / kg per week following IV administration, and in a 26-week repeat-dose toxicity study in cynomolgus monkeys up to 200 mg / kg per week, with no evidence of treatment-related effects on cardiovascular, respiratory, or neurological endpoints, supporting the systemic safety of IVT-administered FabA.

[0326] Additionally, in a 4-week repeat-dose GLP toxicity study in cynomolgus monkeys, safety pharmacology endpoints of FabA were assessed following SC administration up to 20 mg / kg daily, with no evidence of treatment-related effects on cardiovascular, respiratory, or neurological endpoints, supporting the systemic safety of IVT-administered FabA.

[0327] Pharmacokinetics in Animals

[0328] Nonclinical studies designed to characterize the PK, TK, and PD of FabA were conducted in rats and cynomolgus monkeys. These studies included single-dose IVT PK studies in rats and cynomolgus monkeys, and repeated-dose TK / PD studies with FabA in cynomolgus monkeys. More extensive TK / PD studies were conducted in cynomolgus monkeys, and no ocular toxicity was observed in either the rat or cynomolgus monkey single-dose studies.

[0329] Pharmacokinetic / toxicokinetic / pharmacodynamic analysis

[0330] Pharmacokinetics of FabA in vitreous

[0331] Following a single bilateral IVT administration of FabA to rats at either 0.01 mg / eye (equivalent to a 2 mg human dose) or 0.05 mg / eye (equivalent to a 10 mg human dose), the drug was relatively rapidly removed from the vitreous, consistent with a half-life of approximately 12 hours for both dose levels. In cynomolgus monkeys, which also received bilateral IVT FabA, drug distribution from the vitreous was slower compared to rats, with half-lives of approximately 3 days for the 1 mg / eye (equivalent to a 2 mg human dose) and 5 mg / eye (equivalent to a 10 mg human dose) dose groups.

[0332] In both species, FabA IVT PK was dose linear. Data from ocular toxicology studies in cynomolgus monkeys, in which FabA was administered twice over 28 days at 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), showed 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-dose IVT administrations.

[0333] In a 26-week chronic ocular toxicology study in cynomolgus monkeys, in which FabA was administered 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 two weeks (both 5 mg / eye doses were subsequently reduced to 2.5 mg / eye and referred to as 5 / 2.5 mg / eye), vitreous FabA concentrations were quantifiable from Days 184 to 169 in all animals receiving FabA and were below the limit of quantitation (BQL) in all animals on Days 242 / 243 after a 10-week dose-free recovery period. Vitreous FabA concentrations showed a high degree of variability with no clear differences or trends between dose groups or sexes.

[0334] Pharmacokinetics of FabA in serum

[0335] After a single IVT administration, serum concentrations were much lower than those in the vitreous, with C max Serum / Cmax The vitreous concentration was approximately 0.003 in rats and 0.000001 in cynomolgus monkeys. In cynomolgus monkeys that received bilateral FabA IVT twice over a 28-day period, serum concentrations were lower, with the highest mean peak concentration (C max ) was 10.1 ng / mL, which was observed after administration of the second IVT dose of 5 mg / eye (equivalent to a 10 mg human dose). Due to the distribution of FabA from the vitreous to the serum compartment, FabA can bind to C1q, or remain in its free form and can be quantified by the assay, resulting in unquantifiable low FabA serum concentrations (i.e., <1.25 ng / mL) in the 1 mg / eye group, and a mean C of 1.25 ng / mL for 2.5 and 5.0 mg / eye. max 3.3 and 10.1 ng / mL respectively.

[0336] In contrast, after IV administration of FabA at a dose of 10 mg / kg, the maximum concentrations of FabA in the two cynomolgus monkeys tested were 13,800 and 17,000 ng / mL, respectively, after which the concentrations declined very rapidly, consistent with a half-life of approximately 2 hours, as expected for a Fab fragment.

[0337] When serum FabA concentrations following two bilateral IVT administrations (5 mg / eye) over 28 days were compared to those obtained after 4 weeks of systemic IV administration of Mab2 at a dose of 200 mg / kg once weekly, FabA serum exposure was significantly reduced (FabA / Mab2 Cmax ratio of 0.00000701).

[0338] In a 26-week chronic ocular toxicology 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 biweekly, with low systemic exposure of FabA in serum, consistent with a topical route of administration. Serum concentrations of FabA did not exceed 86.3 ng / mL on Day 85, and did not exceed 60.8 ng / mL on Day 169 after the last dose. Maximum serum FabA concentrations were observed 24 to 48 hours post-dose across dose levels / schedules and days evaluated. Half-life (T1 / 2) values ​​for FabA in serum were calculable / reportable in only a few cases for animals in the biweekly 5 / 2.5 mg / eye group and ranged from 49.9 to 143 hours on all evaluation days, likely representing distribution from the ocular space into serum. There was little accumulation of FabA in serum with repeated monthly IVT dosing at 2.5 mg / eye. However, calculable FabA serum concentrations in this group increased with each subsequent assessment day after Day 1. Accumulation in any other group could not be determined from Day 1 due to changes in dose levels after Day 57. The ratio of the area under the curve at Day 169 / Day 85 to time "t" (AUC[0-t]) ranged from 0.0407 to 0.664 in males and females at 5 / 2.5 mg / eye monthly and from 0.132 to 7.15 in males and females at 5 / 2.5 mg / eye biweekly. When comparing the sex-combined systemic exposures of FabA and Mab2 following chronic 26-week dosing in cynomolgus monkeys, FabA serum exposures following biweekly bilateral IVT administration of 5 / 2.5 mg / eye (AUC0-t of 1,230 hr*ng / mL or 1.23 hr*μg / mL) were considerably lower than the 200 mg / kg AUC0-t (3,150,000 hr*μg / mL) obtained following weekly systemic IV administration of Mab2 (FabA / Mab2 C max ratio was 0.000000073 and AUC0-t ratio was 0.00000039).

[0339] Pharmacodynamics of ocular C1q

[0340] In control animals, the mean vitreous free C1q concentration was 40.3 ng / mL, while in the vitreous of cynomolgus monkeys receiving a single FabA IVT dose of 1 mg / eye (equivalent to a 2 mg human dose) or 5 mg / eye (equivalent to a 10 mg human dose), free C1q levels were below the limit of detection (<1.953 ng / mL) for the duration of the study (30 days), indicating that complete C1q inhibition occurred. In cynomolgus monkeys receiving FabA for a total of 2 doses every 28 days, C1q remained inhibited for 15 days after the second dose of all 3 dose levels (i.e., 1, 2.5, and 5 mg / eye q28 days x 2 doses). Thirty days after administration of the second FabA dose, C1q was still below the limit of detection in some but not all eyes.

[0341] Fifteen days after the second 5 mg / eye IVT administration, >80% of C1q was bound to FabA in the retina, choroid, and optic nerve head. Thirty days after the second 5 mg / eye administration, C1q was still inhibited only in the retina and choroid.

[0342] In a 26-week chronic ocular toxicology 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 biweekly. At terminal necropsy, vitreous C1q levels were reduced in all groups receiving FabA; vitreous C1q levels were restored and comparable to controls in animals following a dosing holiday and / or a 10-week no-dose recovery period at terminal and recovery necropsy, respectively.

[0343] Serum C1q and serum hemolysis inhibition

[0344] Following bilateral IVT administration of 5 mg / eye (equivalent to a 10 mg human dose) to cynomolgus monkeys, C1q-dependent serum hemolysis was inhibited by approximately 50-80%, which lasted for approximately 24-48 hours after administration of the first IVT dose and up to 96 hours after administration of the second FabA dose, after which it returned to baseline.

[0345] After a single IV administration of 10 mg / kg to cynomolgus monkeys, maximum inhibition of C1q-dependent serum hemolysis was achieved at 1 hour. Maximum inhibition was maintained for approximately 24 hours and returned to baseline at 120 hours after FabA administration. Serum free C1q also declined rapidly, but had not returned to baseline values ​​by 120 hours, indicating that some FabA remained bound to circulating C1q within this time frame.

[0346] toxicology

[0347] The safety of FabA is supported by a comprehensive nonclinical ocular toxicology program designed to support the use of FabA for IVT administration in clinical trials. Initial single-dose studies were conducted with FabA in rats and cynomolgus monkeys, and no ocular toxicity was observed in either species. Based on similar findings in rats and cynomolgus monkeys, as well as in vitro pharmacology data and sequence homology data (indicating that cynomolgus monkeys are more relevant than rats), cynomolgus monkeys were selected for repeated-dose ocular toxicology studies of FabA.

[0348] Repeated dose ocular toxicology studies included ophthalmological examination (OE), IOP, electroretinogram (ERG), ocular histopathology, and measurement of FabA in serum and vitreous for TK analysis. In addition, FabA PD properties were characterized by measurement of C1q in vitreous (for all repeated dose studies) and ocular tissues (in two-dose studies), and inhibition of C1q-dependent hemolysis in serum (in two-dose studies).

[0349] Single dose toxicity

[0350] IVT administration of FabA was well tolerated in single-dose (rat and cynomolgus monkey) ocular toxicology studies. In these studies, the NOAELs for 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 was the highest dose evaluated in each study and was equivalent (2.5 mg / mL) when corrected for vitreous volume (0.02 mL in rats and 2 mL in cynomolgus monkeys).

[0351] GLP Single-Dose Ocular Toxicity Study of FabA by Intravitreal Injection in Sprague Dawley Rats

[0352] In this single-dose GLP rat ocular toxicology study, vehicle or FabA was administered once bilaterally to young adult male rats 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 terminated on Day 1 (6 hours post-dose), Day 3, Day 7, Day 10, Day 20, Day 30, and all vehicle control animals were terminated on Day 30. All animals survived to scheduled necropsy.

[0353] The study included standard safety parameters. Blood samples were collected at termination and terminal vitreous samples were obtained for TK analysis. In addition, ophthalmological examinations (OE), including IOP and ocular histopathology, were assessed.

[0354] No FabA-related changes were found in any of the safety parameters evaluated, including OE, IOP, and ocular histopathology.

[0355] Vitreous exposure of FabA was confirmed by TK in animals treated at 0.01 mg / eye (equivalent to a 2 mg human dose) and 0.05 mg / eye (equivalent to a 10 mg human dose) from 6 hours (first collection) to 144 hours post-dose. Serum exposure of FabA was confirmed by TK in animals treated at 0.01 and 0.05 mg / eye (2 to 48 hours post-dose only).

[0356] In this study, no adverse reactions considered to be related to Fab A were observed at any dose level, including the highest dose evaluated, 0.05 mg / eye. Based on these results, the NOAEL is 0.05 mg / eye (2.5 mg / mL in the vitreous).

[0357] A non-GLP single-dose ocular toxicity study of FabA by intravitreal injection in cynomolgus monkeys

[0358] In this single-dose non-GLP cynomolgus monkey ocular toxicology study, vehicle or FabA was administered bilaterally by IVT injection to young adult female cynomolgus monkeys at doses of 1 mg / eye (equivalent to 2 mg human dose) and 5 mg / eye (equivalent to 10 mg human dose). FabA-treated animals were terminated on Day 1 (6 hours post-dose), Day 3, Day 7, Day 10, Day 20, Day 30. All vehicle control animals were terminated on Day 30, and all animals survived to scheduled necropsy.

[0359] Standard safety parameters including OE, IOP, and ocular histopathology were assessed in this study. In addition, blood samples were collected throughout the study, and terminal vitreous samples were analyzed for TK and PD.

[0360] FabA-related changes were limited to non-adverse findings not related to inflammation. These findings included histiocytic infiltration in the uvea and mild basophilia in the 1 mg / eye dose group. Results for the 5 mg / eye dose included histiocytic infiltration in the uvea and minimal to mild basophilia.

[0361] No FabA-related changes were observed in OE and IOP. In this study, no adverse reactions considered to be related to FabA were observed at any dose level, 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 in vitreous).

[0362] TK confirmed FabA exposure in the vitreous for all treated animals during the study (until day 30). Serum exposure to FabA was absent 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 by day 30.

[0363] Repeated dose toxicity studies

[0364] In the repeated-dose ocular toxicity studies, FabA was administered at least every 4 weeks via IVT injection. Repeated-dose administration of FabA was well tolerated in cynomolgus monkeys. In the initial repeated-dose GLP ocular toxicology study, the NOAEL in cynomolgus monkeys was 5 mg / eye (equivalent to a 10 mg human dose) once monthly for two doses, the highest dose evaluated. In the 26-week chronic ocular toxicology study in cynomolgus monkeys, adverse ocular changes were related to the dual injection procedure and / or were determined to be ADA-mediated and not a direct effect of FabA IVT administration, so the NOAEL in cynomolgus monkeys was determined to be 2.5 mg / eye (equivalent to a 5 mg human dose) once every two weeks or monthly for 13 or 7 doses, respectively.

[0365] A 6-week GLP repeat-dose ocular toxicity study of FabA by intravitreal injection in cynomolgus monkeys

[0366] The study included standard safety parameters and blood samples were collected throughout the study. Terminal vitreous samples were also collected for TK and PD analysis and terminal optic nerve sections were also collected for TK and PD analysis. In addition, OE, IOP, ERG and ocular histopathology were also evaluated.

[0367] Results for FabA that were determined to be non-adverse events were limited to one high-dose (2.5 mg / eye) (equivalent to a 5 mg human dose) female, which had minimal basophilic / blue staining of the vitreous with no associated inflammation (known as basophilia). Importantly, no FabA-related changes were found in OE, IOP, and ERG. TK confirmed that the vitreous of all treated animals was exposed to FabA during the study and during the recovery period (30 days after the last dose).

[0368] Serum exposure was not measurable at 1 mg / eye (equivalent to a 2 mg human dose), was low and transient at 2.5 mg / eye (equivalent to a 5 mg human dose) (12 to 48 hours after the first dose, 6 to 168 hours after the last dose), and did not exceed 8 ng / mL (LLOQ 1.25 ng / mL). PD confirmed that C1q was absent in the vitreous of all treated animals when FabA levels were approximately 100 ng / mL. FabA ADA was detected in animals at 1 mg / eye (equivalent to a 2 mg human dose) (6 of 12 animals) and 2.5 mg / eye (equivalent to a 5 mg human dose) (7 of 12 animals), but there was no apparent effect on FabA exposure in serum or vitreous by ADA.

[0369] A 6-week GLP repeat-dose ocular toxicity study of FabA by intravitreal injection in cynomolgus monkeys

[0370] In this 6-week GLP cynomolgus ocular toxicology study, vehicle or FabA was administered bilaterally to young adult male and female cynomolgus monkeys at a dose of 5 mg / eye (equivalent to a 10 mg human dose) by IVT injection every four weeks (Days 1 and 29), followed by a 4-week recovery period. All main study animals were terminated on Day 44 and all recovery animals were terminated on Days 59 / 60. All main study and recovery animals survived to scheduled necropsy.

[0371] The study included standard safety parameters (except systemic histopathology), and blood samples were collected throughout the study, as well as terminal vitreous samples for TK and PD analysis. ADA and aqueous humor samples were collected and archived. In addition, OE, IOP, ERG, and ocular histopathology were assessed.

[0372] No FabA-related changes were found in any of the safety parameters evaluated, including OE, IOP, ERG, and ocular histopathology. Minimal to mild basophilic / blue staining of the vitreous (termed basophilia) without associated inflammation was observed in both treated and control animals and was therefore not considered to be related to FabA.

[0373] TK confirmed that the vitreous of all treated animals was exposed to FabA during the study and recovery period (30 days after the last dose). FabA was confirmed to be absent in the serum and vitreous of control animals. PD confirmed that C1q was absent in the vitreous of all treated main study animals at day 44. At day 59, 2 / 4 of the recovery animals had measurable C1q in the vitreous. FabAADA was detected in animals in the 5 mg / eye (equivalent to a 10 mg human dose) group (9 of 10 animals), but there was no apparent effect on FabA exposure in the serum or vitreous of ADA.

[0374] Inhibition of C1q-dependent hemolysis by >80% was achieved 24-48 hours after FabA administration and returned to baseline thereafter.

[0375] C1q levels also decreased significantly at day 44 in the retina, choroid, and optic nerve head, but not in the optic nerve head.

[0376] In this study, no adverse reactions considered to be 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 is 5 mg / eye (equivalent to a 10 mg human dose) (2.5 mg / mL in the vitreous).

[0377] Ocular toxicity of 26-week GLP repeat-dose FabA by intravitreal injection in cynomolgus monkeys with 10-week recovery Research

[0378] In this 26-week GLP cynomolgus ocular toxicology study, vehicle or FabA was administered bilaterally by IVT injection to young adult male and female cynomolgus monkeys at a dose of 2.5 mg / eye once a month (equivalent to a 5 mg human dose), 5 mg / eye once a month (equivalent to a 10 mg human dose), 5 mg / eye every other week (every two weeks), 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) was given every 2 weeks (13 dosing cycles) or every 4 weeks (7 dosing cycles). All main study animals were terminated on day 184 and all recovery animals were terminated on days 242 / 243. All main study and recovery animals survived to scheduled necropsy.

[0379] Dosing holidays or dosing cessation occurred in animals in the control group, 5 mg / eye once a month (equivalent to a 10 mg human dose), and 5 mg / eye once every two weeks dose groups. Due to adverse findings detected by OE, dual injections in these groups were terminated and were considered to be related to the procedure and high dose volume. Starting from day 71 of the study, the 5 mg / eye group once a month was dosed with 2.5 mg / eye (equivalent to a 5 mg human dose) once a month (referred to as 5 / 2.5 mg / eye once a month), and the 5 mg / eye every two weeks was dosed with 2.5 mg / eye every two weeks (referred to as 5 / 2.5 mg / eye every two weeks). After the dual injections were stopped, dosing holidays continued in these dose groups (including the control group), which were related to the procedure and / or ADA, as described below. There was no dosing holiday in the 2.5 mg / eye group once a month (low dose group).

[0380] The study included standard safety parameters (except systemic histopathology), and blood samples were collected throughout the study, as well as terminal vitreous samples for TK and PD analysis. ADA and aqueous humor samples were collected and archived. In addition, OE, IOP, ERG, ocular histopathology, and immunohistochemistry (IHC) for the detection of immune complexes deposited in the spheres were evaluated.

[0381] No FabA-related changes were found in body weight, food consumption, electroretinogram measurements, intraocular pressure measurements, and clinical pathology.

[0382] Clinical ocular signs and ophthalmologic findings thought to be associated with FabA were limited to ocular opacities (possibly due to opacities of the anterior chamber, lens capsule, and / or posterior chamber) and the presence of cells and / or pigment. The presence of these findings in animals without detectable ADA in the serum (4 of 12 Group 2 animals, 2 of 12 Group 3 animals, and 2 of 12 Group 4 animals) suggests an association with FabA. Findings thought to be associated with ADA and possibly related to immune complex deposition tended to be more severe and included the presence of aqueous flare and vitreous opacities, altered pupillary light reflex, and retinal vascular attenuation.

[0383] Following IVT administration of FabA to male and female cynomolgus monkeys, systemic exposure as measured by serum FabA concentrations was transient, low-level, and did not exceed 86.3 ng / mL following dosing on Day 85, or 60.81 ng / mL following the last dose on Day 169. TK demonstrated that exposure to FabA corresponded to undetectable vitreous C1q in nearly all treated animals treated with FabA prior to Day 169, with the exception of some animals with a dosing holiday. Vitreous concentrations of FabA were unmeasurable on Days 242 / 243 following a 10-week no-dose recovery period, and C1q concentrations were detectable in all treated dose groups. FabA was demonstrated to be absent in the serum and vitreous of control animals.

[0384] 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. After day 1, two Group 1 animals were confirmed positive at a single time point per animal, while FabA-treated ADA-positive animals were identified at 3 or 4 or more time points (4 or 5 samples were collected in total for the main study and recovery animals, respectively). ADA had no apparent effect on FabA exposure in serum or vitreous.

[0385] At the time of terminal euthanasia on day 184, microscopic changes consistent with ADA-mediated immune responses to FabA were observed in the right eye at monthly and biweekly 5 / 2.5 mg / eye (mid-dose and high-dose groups, respectively). Intraocular changes associated with inflammation included mild mixed cell infiltration of the ciliary body and vitreous cavity, mild to moderate fibrosis in the vitreous cavity (severity was proportional to the dose frequency), and mild to mild posterior lens degeneration (severity was proportional to the dose frequency). Minimal perivascular mononuclear cell infiltration was also observed in the posterior retina in each female in the biweekly and monthly 5 / 2.5 mg / eye treatment groups. Minimal to mild mononuclear cell infiltration was observed in the periocular limbus of animals administered 5 / 2.5 mg / eye every two weeks and monthly, with severity being proportional to the dose frequency.

[0386] At the recovery euthanasia on days 242 / 243, microscopic changes in the right eye associated with ADA-mediated immune responses to FabA were limited and mild at 5 / 2.5 mg / eye monthly, whereas other changes persisted or developed at 5 / 2.5 mg / eye biweekly. Minimal histiocyte infiltration of the vitreous cavity and uvea and increased basophilia of the vitreous cavity were unique to animals administered FabA 5 / 2.5 mg / eye biweekly and / or monthly at the time of recovery. These changes were similar to those observed in control animals at terminal necropsy, where they were thought to be related to mild inflammation / destruction of the anterior vitreous associated with the IVT injection procedure. However, the persistence of these changes after the recovery period, the resolution of these changes in control animals during the recovery period, and the presence of more severe inflammatory changes at terminal necropsy in animals administered FabA 5 / 2.5 mg / eye biweekly and monthly suggest that these changes at the time of recovery more likely represent resolution of inflammation associated with ADA-mediated immune responses to FabA rather than residual effects of the injection procedure. Minimal mononuclear cell infiltration of the periocular limbus persisted at 5 / 2.5 mg / eye every two weeks and monthly.

[0387] Minimal mixed cellular infiltration and fibrosis of the vitreous cavity persisted at 5 / 2.5 mg / eye every two weeks, while retinal cellularity and hemosiderin pigment were modestly reduced. Minimal perivascular mononuclear infiltration of the retina at the optic disc was observed at 5 / 2.5 mg / eye every two weeks. These changes are also thought to be secondary to ADA-mediated responses to FabA.

[0388] Adverse microscopic changes in pathology are thought to be secondary to ADA-mediated inflammation and include fibrosis within the vitreous cavity, lens degeneration, and loss of retinal cellularity at 5 / 2.5 mg / eye every two weeks and monthly.

[0389] There were no microscopic changes associated with FabA 2.5 mg / eye monthly at the time of terminal or recovery euthanasia.

[0390] Immunohistochemistry was performed on 2 of 12 animals, 4 of 12 animals, and 6 of 12 animals in Groups 1, 3, and 4, respectively. The evaluation showed that granular deposits containing FabA, cynomolgus IgG, IgM, and / or C3 were detected by immunohistochemistry in the left eyes of 4 of the 10 treated animals selected for IHC at the medium dose of 5 / 2.5 mg / eye monthly (2 of 4 animals) and the high dose of 5 / 2.5 mg / eye every two weeks (2 of 6 animals). The presence of these intramural vascular deposits was associated with perivascular inflammatory cell infiltration, similar to those observed by hematoxylin and eosin evaluation of the right eye. Other microscopic changes observed in the right eye were consistent with secondary changes associated with this immune response to FabA in cynomolgus monkeys. Although ocular immune complex deposits were not observed in all animals selected for immunohistochemistry (including some animals that were serum ADA negative), this was not unexpected because deposit identification can vary from tissue section to tissue section, and serum ADA is not always present in animals where microscopic evidence is consistent with immune complex pathology. Additionally, in some animals on multiple dose holidays, ADA and / or immune complexes may have been cleared prior to analysis. The presence of immunohistochemically confirmed deposits, even in a subset of animals, was considered the most compelling evidence that the similar and pathologically consistent pathology observed in the right eye may be related to an immune response to FabA.

[0391] In this 26-week chronic ocular toxicology study in cynomolgus monkeys, adverse ocular changes were determined to be related to the dual injection procedure and / or ADA-mediated and not a direct effect of FabA IVT administration, and thus the NOAEL in cynomolgus monkeys was established at 2.5 mg / eye (equivalent to a 5 mg human dose) every two weeks or monthly, for 13 or 7 doses, respectively.

[0392] Example 9: Evaluation of FabA in clinical studies

[0393] The FabA drug product is a sterile isotonic liquid 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.

[0394] 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.

[0395] Results from both studies found that single doses (1 to 5 mg / eye) (equivalent to a 2-10 mg human dose) and repeated doses (2.5 and 5 mg / eye, 2 doses, 4 weeks apart) of FabA IVT were well tolerated in patients with glaucoma; no serious or significant adverse events (AEs) were reported. In these studies, ocular AEs in patients treated with FabA included conjunctival hyperemia, conjunctival hemorrhage, and eye irritation, and occurred only in the treated eyes. In the Phase 1b study, ocular AEs in patients in the sham injection group included eye pain, foreign body sensation in the eye, ocular hyperemia, and blurred vision. No systemic AEs considered to be related to FabA IVT treatment occurred.

[0396] A single IVT injection of 2.5 mg (equivalent to a 5 mg human dose) and 5 mg (equivalent to a 10 mg human dose) of FabA inhibited free C1q in the aqueous humor for at least 29 days (Study FabA-GLA-02).

[0397] Human Pharmacokinetics and Pharmacodynamics

[0398] Ocular Pharmacokinetics and Pharmacodynamics

[0399] FabA-GLA-02 is a Phase 1b study in which the aqueous humor is sampled to assess PK and PD. Subjects were administered two IVT sham injections of either 2.5 mg / eye FabA (equivalent to a 5 mg human dose) or 5 mg / eye (equivalent to a 10 mg human dose) FabA, 29 days apart. In this study, the aqueous humor was sampled pre-dose and 29 days after the first FabA dose and before the second dose. Free FabA was detected in the aqueous humor of all treated patients on Day 29 (D29). In parallel, FabA at dose levels of 2.5 mg / eye and 5 mg / eye inhibited free C1q in the aqueous humor for at least 29 days ( Fig.12 ).

[0400] Systemic pharmacokinetics and pharmacodynamics

[0401] FabA-GLA-01 was a single-dose Phase 1 study in which serum FabA and C1q were sampled before and 3 hours after dosing. FabA-GLA-02 was a multiple-dose Phase 1b study in which serum and FabA and C1q were sampled before and 3 hours after each of 2 doses separated by 29 days. FabA was generally undetectable in the systemic circulation following single or repeated IVT injections at any dose level studied in the Phase 1 or Phase 1b clinical studies. Similarly, no changes in circulating free C1q were detected in either of these two studies.

[0402] As described below, in the FabA clinical study, the 5 mg / eye (equivalent to a 10 mg human dose) dose level was well tolerated as a single dose or as two doses 29 days apart. As described above, in the Phase 1b study, single doses of FabA at 2.5 mg (equivalent to a 5 mg human dose) and 5 mg (equivalent to a 10 mg human dose) inhibited free C1q in the aqueous humor for at least 29 days ( Fig.12 ).

[0403] Safety and efficacy

[0404] Phase 1 Dose Escalation (FabA-GLA-01)

[0405] This is 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 with a mean deviation of 3 to 18 dB on reliable visual field testing, who were able to perform reliable visual field testing in the study eye, a cutoff of 33% for fixation loss using the Humphrey Field Analyzer-Swedish Interactive Threshold Algorithm (HFA-SITA) 24-2 fast algorithm, a false-positive response rate of 33%, and an IOP <21 mmHg in the study eye on a stable IOP treatment regimen for ≥4 weeks prior to dosing. Nine patients were assigned to 3 cohorts of 3 patients each, as follows:

[0406] Group 1 = 1.0 mg / eye, single dose (0.02 mL) x 1 dose

[0407] Group 2 = 2.5 mg / eye, single dose (0.05 mL) × 1 dose

[0408] Group 3 = 5.0 mg / eye, single dose (0.10 mL) × 1 dose

[0409] After screening, 3 eligible patients were enrolled in the lowest open group, and the next group was enrolled only after tolerability and short-term safety at the previous lower dose were confirmed. All patients in each group were required to complete a safety observation period of at least 15 days before the next group of patients could be injected. No dose-limiting toxicity (DLT) was reported during the study.

[0410] Nine patients were enrolled, treated, and completed the study.

[0411] Security

[0412] Ocular treatment-emergent adverse events (TEAEs) included conjunctival hyperemia (all dose levels), conjunctival hemorrhage (2.5 mg / eye only), and eye irritation (1 mg / eye only) and occurred only in the study eye.

[0413] The only systemic TEAE experienced in the study was sinusitis.

[0414] All TEAEs were mild in severity.

[0415] There were no serious or significant TEAEs.

[0416] No patient discontinued treatment or withdrew from the study due to TEAEs.

[0417] · Nine of nine patients had normal IOP within 30 minutes (within 5 mmHg of the immediate pre-injection IOP or <21 mmHg).

[0418] • No patient showed any evidence of anti-FabA antibodies.

[0419] Overall summary / conclusion:

[0420] In this study of patients with stable glaucoma, single IVT doses of FabA were well tolerated up to 5 mg / eye. Ocular AEs reported were similar to those reported with IVT administration of approved drugs. No safety signals were observed for FabA.

[0421] FabA was generally undetectable in the systemic circulation, and no changes in circulating free C1q were detected after a single IVT administration.

[0422] Phase 1b (FabA-GLA-02)

[0423] This was a double-blind, randomized, sham-controlled study evaluating two dose levels of FabA administered as repeat IVT injections in patients with primary open-angle glaucoma. Eligible patients were adults with a mean deviation of -3 to -24 dB on reliable visual field testing in the study eye, who were able to perform reliable visual field testing in the study eye, a cutoff of 33% for fixation loss using the HFA-SITA fast algorithm, a false-positive response rate of 33%, IOP <21 mmHg at screening and Day 1, and no expected change in IOP treatment regimen during the study on a stable IOP regimen for ≥4 weeks prior to injection. Patients received two injections, 4 weeks apart, and were followed up for a total of 12 weeks to assess safety, tolerability, PK, PD, immunogenicity, and ongoing exploratory assessments. Patients were randomized (1:1:1) to one of 3 cohorts (5 patients planned per cohort) as follows:

[0424] Dose level 1 = 2.5 mg / eye, single dose (0.05 mL) x 2 doses

[0425] Dose level 2 = 5.0 mg / eye, single dose (0.10 mL) x 2 doses

[0426] Sham injection = 0 mg / eye x 2 doses

[0427] Eighteen patients were randomized (7 to 2.5 mg FabA, 5 to 5.0 mg FabA, and 6 to sham injection), and 17 patients were treated. One patient in the 2.5 mg dose group was randomized but did not receive treatment. Sixteen patients completed the study.

[0428] Security

[0429] 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); no patients in the sham group experienced these TEAEs. Ocular TEAEs in the sham group included eye pain, foreign body sensation in the eye, ocular hyperemia, and blurred vision in one patient each.

[0430] Systemic TEAEs occurred during the study; none were considered by the investigator to be related to study treatment.

[0431] All TEAEs were mild in severity.

[0432] All but one of the reported TEAEs occurred after the first dose but before the second administration of study treatment.

[0433] There were no serious or significant TEAEs.

[0434] No patient discontinued treatment or withdrew from the study due to TEAEs.

[0435] IOP returned to normal (<21 mmHg) within 30 minutes after IVT injection in 16 of 17 patients and within 45 minutes in the remaining patients.

[0436] Of the 11 patients who were given FabA intravitreally, 6 tested positive at at least one time point. One patient was ADA positive with a modest increase in titer over time, and the remaining 5 patients were positive at all time points (including pre-dose) without any change in titer over time. The one sham-injected patient was ADA positive at all time points (including pre-dose) without any change in titer over time. Overall, these data suggest that the relationship between ADA measurements and FabA administration is unclear.

[0437] Overall summary / conclusion:

[0438] In patients with stable glaucoma, 2 IVT doses of FabA, 4 weeks apart, were well tolerated up to 5 mg / eye. Ocular AEs reported were similar to those reported with IVT administration of approved drugs. No safety signals were observed for FabA in this study.

[0439] A single dose of FabA IVT (2.5 and 5 mg / eye) inhibited free C1q in the aqueous humor for at least 29 days.

[0440] Example 10: Vitreous retinal foveation in patients with geographic atrophy (GA) secondary to age-related macular degeneration (AMD) A phase 2, multicenter, randomized, parallel-group, double-blind, 4-group, sham-controlled study of the efficacy, safety, and tolerability of FabA administered by injection in vivo Photo Study

[0441] Basic principle:

[0442] Brief Overview:

[0443] This study is being conducted in patients with GA secondary to AMD. The aim of the study is to determine whether intravitreal (IVT) injections of FabA given monthly (EM) or every other month (EOM) for 12 months can reduce the growth rate of GA lesions. The study consists of a 30-day screening period and a 12-month treatment period, followed by a 6-month (off-treatment) follow-up period. The total duration of patient participation is 19 months. During the 12-month treatment period, patients will visit the clinic every month for treatment and / or safety assessments.

[0444] Approximately 240 patients were enrolled and randomly assigned to 1 of the 4 treatment groups, so approximately 204 patients were evaluable for the primary analysis at month 12 (the primary analysis was based on modified intention-to-treat [ITT]).

[0445] Intervention Groups and Duration:

[0446] Study intervention allocation was based on randomization (2:2:1:1). Patients were assigned to one of the following treatment arms. Dose levels were fixed and would not be modified.

[0447] Group 1 = FabA 5.0 mg / eye (0.10 mL), once a month (EM), for 12 months (12 doses)

[0448] Group 2 = FabA 5.0 mg / eye (0.10 mL), every other month (EOM), for 12 months (6 doses)

[0449] · Experimental group 3 = sham EM injection for 12 months (12 sham injections)

[0450] · Experimental Group 4 = Sham injection of EOM 12 months (6 sham injections)

[0451] injection

[0452] FabA / sham injection administration was performed by the injecting physician using aseptic technique.

[0453] All patients randomized to FabA received 5.0 mg / eye IVT (in a fixed volume of 0.10 mL) either monthly or every other month for 12 months.

[0454] After injection

[0455] After drug administration, the injecting physician immediately assessed manual vision or visualization of central retinal artery perfusion. Other causes of visual loss, such as vitreous hemorrhage, were excluded if necessary. Digital massage was performed if necessary, and topical / oral IOP-lowering medications were administered until manual vision or central retinal artery perfusion was observed.

[0456] IOP (intraocular pressure measurement) was assessed in the study eye only 30 minutes after drug administration and, if elevated, every 15 minutes thereafter until IOP < 25 mmHg.

[0457] Pharmacokinetics, pharmacodynamics, and immunogenicity

[0458] Blood samples for PK (serum concentration of FabA) and PD assessments (serum concentration of C1q and plasma concentrations of other biomarkers) were collected at the visit within 30 minutes before and within 3 hours (±15 minutes) after dosing.

[0459] Samples for immunogenicity testing (ADA) were collected prior to injection during the site visit. Additionally, ADA samples were collected at week 2 at the site or home health visit.

[0460] Pharmacokinetics: This test requires serum. Blood samples are collected for measurement of serum concentrations of FabA.

[0461] Pharmacodynamics: This test requires serum and plasma. Serum concentrations of C1q and plasma concentrations of exploratory complement biomarkers are analyzed.

[0462] Immunogenicity: This test requires serum. Immunogenicity is assessed by analysis of serum anti-drug (FabA) antibodies (ADA).

[0463] Intravitreal injection procedure

[0464] Preparation of FabA

[0465] Using a sterile 1.0 cc syringe with a 19 gauge x 1-1 / 2 inch, 5 micron filter needle, withdraw the entire volume of FabA (approximately 0.3 mL) from the sterile vial of FabA.

[0466] Replace the filter needle with a 30 gauge x 1 / 2 inch injection needle. Excess FabA volume was expelled from the syringe prior to injection, leaving only the required injection volume in the syringe.

[0467] The dose volume of FabA was fixed at 0.10 mL, administered monthly (EM) for 12 months (12 doses) or every other month (EOM) for 12 months (6 doses).

[0468] Preparation for intravitreal injection

[0469] 1. Verify the study eye.

[0470] 2. Measure and record the preoperative intraocular pressure (IOP) of the study eye prior to injection. Perform IOP measurements on the study eye only. IOP must be ≤21 mmHg to proceed. If >21 mmHg, reschedule the injection of FabA and manage IOP at the investigator's discretion.

[0471] 3. 30 minutes before injection, apply one drop of 2.5% phenylephrine hydrochloride eye drops topically to the study eye to allow visualization of the posterior pole after injection (if necessary).

[0472] 4. Before injection:

[0473] Have the patient lie supine on an examination chair with the neck well supported.

[0474] Intravitreal injection

[0475] 1. Wash your hands, wear sterile gloves and a surgical mask when injecting.

[0476] 2. 0.5% proparacaine was applied topically to the study eye.

[0477] 3. Apply 10% povidone-iodine to the eyelashes and eyelid margins. Avoid excessive eyelid massage before or after injection to avoid meibomian gland expression.

[0478] 4. Retract the eyelids from the intended injection site during the procedure. The use of an eyelid speculum is recommended.

[0479] 5. Apply 5% povidone-iodine to the conjunctival surface, including the intended injection site.

[0480] 6. For FabA injection: Insert the needle perpendicular to the sclera, 3.5 to 4 mm posterior to the limbus, between the vertical and horizontal rectus muscles. Apply a sterile cotton-tipped applicator to the injection site immediately after needle removal to reduce vitreous reflux.

[0481] 7. Sham injection: Preparation and post-injection care for sham injections are the same as for Fab A injections. Sham injections are performed by applying pressure to the eye at the typical intravitreal injection site using the blunt end of an empty syringe without a needle.

[0482] After intravitreal injection

[0483] 1. Patients should remain in the clinic for ocular assessment and safety follow-up after injection.

[0484] 2. Immediately assess manual vision or central retinal artery perfusion and exclude other causes of visual loss, such as vitreous hemorrhage. If no other causes are found, perform digital massage and apply topical / oral IOP-lowering medications until manual vision or central retinal artery perfusion is observed.

[0485] 3. IOP measurements were performed on the study eye only 30 minutes after injection and, if elevated, every 15 minutes until IOP < 25 mmHg. Intraocular pressure exceeding 30 mmHg for more than 15 minutes should be treated at the physician's discretion.

[0486] 4. No topical antibiotics are required.

[0487] Example 11: C1q mediates microglial activation of photoreceptor synapses in a light-induced damage model of photoreceptor degeneration prune

[0488] In a light damage model, anti-C1q treatment (e.g., Mab3) reduces retinal complement levels ( Figure 5 A-5D), reduce inflammation ( Fig.13A and Fig. 13B ) and reduce neurodegeneration ( Fig.13A and Figures 13C-13D ). Figures 13A-13D Immunofluorescence (IF) data are shown. Fig. 13B Shown is reduced microgliosis in the outer plexiform layer (OPL) (also called outer synaptic layer) of the retina at day 3 after treatment. The reduction in microgliosis is associated with reduced inflammation. Fig. 13C showed remarkable preservation of photoreceptor synapses, and Fig.13D Significant preservation of cell bodies was demonstrated at day 5 post-treatment. Measurement of preserved photoreceptor synapses and cell bodies following treatment demonstrated that treatment with anti-C1q reduced neurodegeneration in this light damage model.

[0489] Example 12: Anti-C1q intravitreal treatment reduces retinal complement component levels in the rd10 mouse model

[0490] Measurable levels of treatment (e.g., Mab3) were found in retinal lysates from animals treated with anti-C1q, but not in IgG1-treated or untreated groups ( Fig.14A Increased levels of the classical complement component C1q were demonstrated in retinal lysates from untreated and IgG d10 animals compared to WT ( Fig. 14B Anti-C1q treatment resulted in decreased C1q levels in retinal lysates compared with the IgG1-treated and untreated rd10 groups ( Fig. 14B ), demonstrating good measurable PK and C1q binding in the retina (and in plasma, data not shown). Fig.15A is a bar graph representing the quantification of immunofluorescence images, and Fig. 15B Shown are immunofluorescence images demonstrating preservation of photoreceptor synapses (BSN marker) after treatment with a C1q inhibitor. This is evidence of photoreceptor synapse protection.

[0491] Incorporated by Reference

[0492] Each of the patents, published patent applications, and non-patent references cited herein is hereby incorporated by reference in its entirety.

[0493] Equivalent solutions

[0494] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.

Claims

1. A method of treating an inherited retinal disease in a human patient comprising administering to the patient by intravitreal injection a composition comprising about 1 mg to about 10 mg of an anti-C1q antibody, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising HVR-L1 having an amino acid sequence of SEQ ID NO:5, HVR-L2 having an amino acid of SEQ ID NO:6, and HVR-L3 having an amino acid of SEQ ID NO:7; the heavy chain variable domain comprising HVR-H1 having an amino acid sequence of SEQ ID NO:9, HVR-H2 having an amino acid of SEQ ID NO:10, and HVR-H3 having an amino acid of SEQ ID NO:

11.

2. The method of claim 1, wherein the inherited retinal disease is retinitis pigmentosa.

3. The method of claim 1, wherein the inherited retinal disease is choroidal coloboma.

4. The method of claim 1, wherein the inherited retinal disease is fundus yellow spot disease.

5. The method of claim 1, wherein the inherited retinal disease is cone-rod dystrophy.

6. The method of claim 1, wherein the inherited retinal disease is Leber congenital amaurosis.

7. The method of claim 1, wherein the inherited retinal disease is X-linked RP.

8. The method of claim 1, wherein the inherited retinal disease is Usher's syndrome.

9. A method of treating retinal detachment in a human patient comprising administering to the patient by intravitreal injection a composition comprising about 1 mg to about 10 mg of an anti-C1q antibody, wherein the antibody comprises a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising HVR-L1 having an amino acid sequence of SEQ ID NO:5, HVR-L2 having an amino acid of SEQ ID NO:6, and HVR-L3 having an amino acid of SEQ ID NO:7; the heavy chain variable domain comprising HVR-H1 having an amino acid sequence of SEQ ID NO:9, HVR-H2 having an amino acid of SEQ ID NO:10, and HVR-H3 having an amino acid of SEQ ID NO:

11.

10. The method of claim 9, wherein the anti-Clq antibody is administered prior to retinal detachment surgery.

11. The method of claim 9, wherein the anti-C1q antibody is administered following retinal detachment surgery.

12. The method of claim 9, wherein the anti-C1q antibody is administered concurrently with retinal detachment surgery.

13. The method of any one of claims 1 to 12, wherein the method restores vision to the human patient.

14. The method of any one of claims 1 to 12, wherein the method improves the vision of the human patient.

15. The method of any one of claims 1 to 14, wherein the antibody comprises a light chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 4 and 35-38, and wherein the light chain variable domain comprises HVR-L1 having an amino acid sequence of SEQ ID NO: 5, HVR-L2 having amino acids of SEQ ID NO: 6, and HVR-L3 having amino acids of SEQ ID NO:

7.

16. The method of claim 15, wherein the light chain variable domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 35-38.

17. The method of any one of claims 1 to 16, wherein the antibody comprises a heavy chain variable domain comprising an amino acid sequence having at least about 95% homology to an amino acid sequence selected from SEQ ID NOs: 8 and 31-34, and wherein the heavy chain variable domain comprises an HVR-H1 having an amino acid sequence of SEQ ID NO: 9, an HVR-H2 having amino acids of SEQ ID NO: 10, and an HVR-H3 having amino acids of SEQ ID NO:

11.

18. The method of claim 17, wherein the heavy chain variable domain comprises an amino acid sequence selected from SEQ ID NOs: 8 and 31 to 34.

19. The method of any one of claims 1 to 18, wherein the antibody is a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, an antibody fragment, or an antibody derivative thereof.

20. The method of claim 19, wherein the antibody is an antibody fragment, and the antibody fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a diabody or a single-chain antibody molecule.

21. The method of claim 20, wherein 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.

22. The method of any one of claims 1 to 21, wherein the antibody is administered once a week.

23. The method of any one of claims 1 to 21, wherein the antibody is administered every other week.

24. The method of any one of claims 1 to 21, wherein the antibody is administered once every three weeks.

25. The method of any one of claims 1 to 21, wherein the antibody is administered monthly.

26. The method of any one of claims 1 to 21, wherein the antibody is administered once every four weeks.

27. The method of any one of claims 1 to 21, wherein the antibody is administered once every six weeks.

28. The method of any one of claims 1 to 21, wherein the antibody is administered once every 8 weeks.

29. The method of any one of claims 1 to 21, wherein the antibody is administered every other month.

30. The method of any one of claims 1 to 21, wherein the antibody is administered once every 10 weeks.

31. The method of any one of claims 1 to 21, wherein the antibody is administered once every 12 weeks.

32. The method of any one of claims 1 to 21, wherein the antibody is administered once every three months.

33. The method of any one of claims 1 to 21, wherein the antibody is administered once every 4 months.

34. The method of any one of claims 22 to 33, wherein the antibody is administered for at least 3 months, at least 4 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months.

35. The method of any one of claims 22 to 34, wherein the antibody is administered for 12 months.

36. The method of any one of claims 1 to 35, wherein 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 the anti-C1q antibody.

37. The method of any one of claims 1 to 36, wherein the composition comprises administering about 1 mg of the anti-C1q antibody.

38. The method of any one of claims 1 to 36, wherein the composition comprises administering about 2.5 mg of the anti-C1q antibody.

39. The method of any one of claims 1 to 36, wherein the composition comprises administering about 5 mg of the anti-C1q antibody.

40. The method of any one of claims 1 to 36, wherein the composition comprises administering about 2 mg of the anti-C1q antibody.

41. The method of any one of claims 1 to 36, wherein the composition comprises administering about 5 mg of the anti-C1q antibody.

42. The method of any one of claims 1 to 36, wherein the composition comprises administering about 10 mg of the anti-C1q antibody.

43. The method of any one of claims 1 to 36, wherein the composition comprises administering 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 the anti-C1q antibody.

Images