Anti-C3 antibodies and antigen-binding fragments thereof and their use for treating ophthalmic or ocular diseases - Patents.com

Abstract

The present invention relates to antibodies and fragments thereof that target complement C3. More specifically, anti-C3 antibodies and methods of use for the treatment of various diseases or disorders are disclosed.

Description

[Technical field]

[0001] The present invention generally relates to antibodies and fragments thereof that target complement C3. More specifically, anti-C3 antibodies and antigen-binding fragments thereof and methods of use for treating various diseases or disorders are disclosed. Pharmaceutical compositions comprising anti-C3 antibodies are also disclosed. [Background technology]

[0002] Age-related macular degeneration (AMD) is generally classified into two main classes: dry AMD and wet AMD. Dry AMD is also known as non-exudative AMD and is characterized by the presence of drusen (yellow deposits) in the macular region. Wet AMD is also known as exudative AMD or neovascular AMD and is characterized by the growth of abnormal blood vessels from the choroid under the macula. This process is also called choroidal neovascularization, and the new blood vessels may leak fluids such as blood into and around the retina. Geographic atrophy (GA), also known as atrophic AMD or advanced atrophic AMD, is a progressive form of AMD characterized by loss of retinal pigment epithelium (RPE) and photoreceptors in the macula. When GA affects the fovea, it causes irreversible vision loss. Patients with early stage GA typically experience loss of visual function before vision is affected. The pathophysiology underlying geographic atrophy is not fully understood; however, dysregulation of complement activity is thought to be a contributing factor. Several complement activation products, including C3a, C5a, C5b-9, and complement factor H (CFH), have shown elevated levels in vitreous samples, Bruch's membrane, and other parts of the choroid of GA patients compared to controls. In addition, complement inhibitors such as CD59 (a membrane-bound inhibitor of membrane attack complex (MAC) formation) and membrane cofactor proteins (MCPs), membrane-bound complement regulators with cofactor activity for complement factor I (CFI) have been reported to have reduced levels in GA. One major challenge in the treatment of GA is that the observed dysregulation of complement activity occurs in the deep layers of the retina. Currently, there are no adequate treatments for GA with acceptable efficacy and patient acceptability and compliance. There remains an unmet need for new and improved therapeutic approaches to effectively treat ophthalmic or eye diseases such as geographic atrophy, and to restore vision or prevent the decline of vision in patients suffering from such diseases. Summary of the Invention

[0003] The innate human system is composed of the complement pathway. It bridges innate and adaptive immunity; it disposes of immune complexes and products of inflammatory injury, and acts as a defense against pyogenic bacterial infections. Complement is a system involving more than 30 proteins in a cascade reaction in plasma and on the cell surface. The complement system and its complement components are involved in various immune processes. For example, the complement C5b-9 complex, also called the terminal complex or membrane attack complex (MAC), plays a key role in cell death by inducing membrane permeability damage. There are three known activation pathways: classical, lectin and alternative. All three pathways result in the cleavage of C3 by C3 convertase and subsequent cleavage of C5 by C5 convertase, releasing C3a, C3b, C5a and C5b. Complement C3 is a large protein composed of 13 distinct domains with a molecular size of 185 kilodaltons. During complement activation, C3 undergoes proteolytic cleavage at various sites and structural modifications. C3-derived fragments exert various effector functions and form convertases that stimulate amplification loops to the three complement pathways. For the avoidance of doubt, unless otherwise indicated, as used herein, C3 refers to human complement component 3 and the nucleic acid sequence encoding the protein in UniProt P01024. The sequence of human complement C3 is shown in SEQ ID NO: 47 and is available online at UniProt P01024.

[0004] C3b is derived from native C3 and is the larger of two components formed by cleavage of C3. The classical and lectin pathway C3 convertases, C4bC2a, cleave full-length C3 into C3b and the anaphylatoxin C3a. The alternative pathway also produces C3b and C3a, but utilizes the alternative pathway C3 convertase, C3bBb. Additionally, additional C3 degradation products can be generated in the complement pathway. Complement factor I (CFI) is a plasma serine protease that can persistently inactivate C3b to iC3b. iC3b is then cleaved by CFI into further fragments (C3dg and C3c). An additional C3 proteolytic product, C3d, binds to complement receptor 2 (CR2) and may play an important role in cell cycle control of B cells. Along with C3 derived protein products, the complement pathway includes, but is not limited to, C1, C2, C4, C4b, C4a, C5, C5b, C5a, C6, C7, C8, C9, C1q, C1r, C1s, Factor B, Factor D, Factor P, Factor H, Factor I, CD46 (MCP), CD55 (DAF), CD59 (MAC-IP), CR1 (CD35), CR2 (CD21), CR3, CR4, C3aR, C5aR1, C5aR2, CRIg, C4BP alpha chain, C4BP beta chain, ficolin-1, mannose-binding lectin (MBL), MBL-associated serine protease-1 (MASP-1), and MBL-associated serine protease-2 (MASP-2). The complement pathway and various complement pathway components are described in further detail in Noris et al. Semin Nephrol. 2013; 33(6): 479-492.

[0005] Recent studies have demonstrated that complement components C3 and C5 are major constituents of drusen in patients with AMD. Their presence in the RPE cells overlying drusen, as well as the presence of the membrane attack complex (MAC) C5b-9 and other acute phase reactant proteins, is speculated to be involved in processes that may lead to complement activation and the formation of the MAC. It has been shown that in patients with GA, increased and uncontrolled activation of the complement system in the choroid and retina leads to destruction of choriocapillaris, damage and loss of both RPE cells and photoreceptors. We hypothesize and demonstrate that neutralization of C3 in the retina blocks the amplification loop of the alternative complement pathway, thereby reducing the production of cytotoxic membrane attack complex and proinflammatory complement components (C3a, C3b, iC3b, C5a), improving clinical outcomes in patients with GA. However, treating ophthalmic or ocular diseases is particularly challenging because several barriers, including but not limited to the blood-retinal barrier, limit the delivery of therapeutic agents to the eye. The ability to penetrate the RPE and enter the choroid of the eye enhances the therapeutic potential of the drug. To address this clinical situation, the present inventors have developed novel antibodies and fragments thereof that can penetrate the RPE and Bruch's membrane in the choroidal region of the eye, thereby targeting complement C3 in the choroidal region. The antibodies and fragments thereof of the present invention exhibit enhanced properties that improve clinical outcomes in patients suffering from ophthalmic or ocular diseases.

[0006] In one aspect, the invention provides an anti-C3 antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) and a variable light chain (VL): - the VH comprises the CDR-H1 sequence of SEQ ID NO: 1, a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2 and 15, and a CDR-H3 sequence of SEQ ID NO: 3; and - the VL comprises a CDR-L1 sequence of SEQ ID NO: 4, a CDR-L2 sequence selected from the group consisting of SEQ ID NO: 5 and 18, and a CDR-L3 sequence of SEQ ID NO: 6; An anti-C3 antibody or an antigen-binding fragment thereof is provided. In one embodiment, the present invention provides - a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 20, 22 or 24; and - a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 21, 23 or 25 The present invention provides an anti-C3 antibody or an antigen-binding fragment thereof, comprising:

[0007] In another embodiment, the present invention provides - a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 20, 22 or 24; and - a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 21, 23 or 25 An anti-C3 antibody or an antigen-binding fragment thereof comprising: - the VH comprises the CDR-H1 sequence of SEQ ID NO: 1, a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2 and 15, and a CDR-H3 sequence of SEQ ID NO: 3; and - the VL comprises a CDR-L1 sequence of SEQ ID NO: 4, a CDR-L2 sequence selected from the group consisting of SEQ ID NO: 5 and 18, and a CDR-L3 sequence of SEQ ID NO: 6; An anti-C3 antibody or an antigen-binding fragment thereof is provided.

[0008] In another embodiment, the present invention provides - a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 20; and - a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 21 An anti-C3 antibody or an antigen-binding fragment thereof comprising: - the VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 2 and the CDR-H3 sequence of SEQ ID NO: 3; and - the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6; An anti-C3 antibody or an antigen-binding fragment thereof is provided.

[0009] In another embodiment, the present invention provides - a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 22; and - a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 23 An anti-C3 antibody or an antigen-binding fragment thereof comprising: - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 15 and the CDR-H3 sequence of SEQ ID NO: 3; and VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6; An anti-C3 antibody or an antigen-binding fragment thereof is provided. In another embodiment, the present invention provides - a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 24; and - a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 25 An anti-C3 antibody or an antigen-binding fragment thereof comprising: - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 15 and the CDR-H3 sequence of SEQ ID NO: 3; and VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 18 and the CDR-L3 sequence of SEQ ID NO: 6; An anti-C3 antibody or an antigen-binding fragment thereof is provided.

[0010] In one embodiment, the invention provides an anti-C3 antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20, 22 or 24; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21, 23 or 25. In a further embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: a. a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO:20 and SEQ ID NO:21, respectively; b. a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO:22 and SEQ ID NO:23, respectively; or c. A variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:25, respectively. The present invention provides an anti-C3 antibody or an antigen-binding fragment thereof, comprising: In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention is selected from the group consisting of a single chain variable fragment (scFv), a Fab fragment, a Fab' fragment, an Fv fragment, a diabody, and a small antibody mimetic. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention is a single chain variable fragment (scFv) comprising or consisting of a variable heavy chain and a variable light chain linked by a polypeptide linker. The scFv may comprise or consist of, from N-terminus to C-terminus, a variable light chain, a linker and a variable heavy chain. The scFv may comprise or consist of, from N-terminus to C-terminus, a variable heavy chain, a linker and a variable light chain. The linker may comprise a polypeptide selected from SEQ ID NOs: 43 to 46 and 56 to 65, preferably SEQ ID NO: 46.

[0011] In an alternative embodiment, the anti-C3 antibody or antigen-binding fragment thereof is selected from the group consisting of single domain antibodies, such as sdAb, sdFv, nanobody, V-Nar and VHH. In this particular embodiment, the anti-C3 antibody or antigen-binding fragment thereof comprises a variable heavy chain. In one embodiment, the variable heavy chain comprises a CDR-H1 sequence of SEQ ID NO: 1, a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2 and 15, and a CDR-H3 sequence of SEQ ID NO: 3. In another embodiment, the variable heavy chain comprises the amino acid sequence of SEQ ID NO: 20, 22 or 24. In one embodiment, said antigen-binding fragment is a single chain variable fragment (scFv), more preferably a humanized scFv. In a further embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention is a single-chain variable fragment comprising a polypeptide having at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30 and 31. In a further embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention is a single-chain variable fragment comprising one sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30 and 31.

[0012] In a further embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention is a single-chain variable fragment consisting of one sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30 and 31. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention can inhibit the pathways of complement activation, including the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP). In certain embodiments, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention can inhibit the activity of the CP, LP and AP complement pathways approximately equally. For example, but not limited to, the anti-C3 antibody or antigen-binding fragment thereof can inhibit the activity of the CP pathway by at least 80%, the activity of the LP by at least 80%, and the activity of the AP by at least 80%. In certain embodiments, the inhibition of the activity of the CP, LP and AP complement pathways is at least 80%, at least 85%, at least about 90% or at least about 95%. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention is capable of binding to complements C3 and C3b.

[0013] In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention may prevent the formation of C3 convertase convertase. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention is capable of penetrating Bruch's membrane. In a specific embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention binds to human C3 and has a K D <50 nM, preferably K D <15 nM, preferably K D <10 nM, preferably K D <7 nM, preferably K D <1 nM, preferably K D <0.5 nM, preferably K D <0.2 nM, preferably K D <0.15 nM, preferably K D <0.10 nM, preferably K D <0.05 nM, preferably K D <0.04 nM, more preferably K D Binds at <0.03nM. In another embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention binds to human C3b with a K D <50 nM, preferably K D <15 nM, preferably K D <10 nM, preferably K D <7 nM, preferably K D <1 nM, preferably K D <0.5 nM, preferably K D <0.2 nM, preferably K D <0.15 nM, preferably K D <0.10 nM, more preferably K D Binds at <0.05 nM.

[0014] In one embodiment, an anti-C3 antibody or antigen-binding fragment thereof according to the invention has approximately equal binding affinity for C3 and C3b. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention has an activity against C3a, iC3b, C4, C4b, C5 and / or C5b of about 10 -4 It has a binding affinity equal to or less than M (i.e., weak). In a further embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention has a weaker binding affinity for C3a, iC3b, C4, C4b, C5 and / or C5b compared to its binding affinity for C3 and C3b. In certain embodiments, the anti-C3 antibodies or antigen-binding fragments thereof according to the invention have no binding affinity for C3a, iC3b, C4, C4b, C5 and / or C5b. As used herein, "no binding affinity" refers to no detectable binding affinity compared to background using one or more binding affinity assays known in the art, such as, but not limited to, an ELISA assay. In certain embodiments, the antibodies of the present invention can bind to complement C3 in a manner that prevents the formation of C3 convertase. In certain embodiments, the antibodies of the present invention can inhibit the activity of C3 convertase. In certain embodiments, the antibodies of the present invention can inhibit the C3 convertase amplification loop.

[0015] In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention can inhibit choroidal C3 activity. In certain embodiments, the anti-C3 antibodies of the present invention are expected to have better efficacy and safety in treating GA or other ophthalmic or eye diseases compared to other therapies due to the properties listed below. In a preferred embodiment, an anti-C3 antibody or antigen-binding fragment thereof according to the invention is capable of binding to an epitope on complement C3, such binding preventing the formation of C3 convertase. In one embodiment, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within the amino acid region set forth in SEQ ID NO: 47. In a preferred embodiment, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof that binds to the amino acid region set forth in SEQ ID NO: 48. In one aspect, the present invention provides an anti-C3 antibody or an antigen-binding fragment thereof that binds to at least one amino acid residue within amino acid 366 to amino acid 478 of human complement C3 set forth in SEQ ID NO:47.

[0016] In one embodiment, the present invention provides an anti-C3 antibody or an antigen-binding fragment thereof that binds to at least one amino acid residue selected from the group consisting of residues 366, 392 to 396, 413 to 421, 425, 427, 442, 453, and 478 of human complement C3 set forth in SEQ ID NO: 47. In one embodiment, the present invention provides an anti-C3 antibody or an antigen-binding fragment thereof that binds to all of amino acid residues 366, 392 to 396, 413 to 421, 425, 427, 442, 453, and 478 of human complement C3 set forth in SEQ ID NO:47. In one aspect, the invention provides an anti-C3 antibody, or an antigen-binding fragment thereof, for use as a medicament. In one embodiment, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof for use in the treatment or prevention of a retinal or ophthalmic disease. Preferably, said retinal or ophthalmic disease is a complement C3-mediated disease or disorder. More preferably, said disease refers to any disorder in which the onset, progression or persistence of the symptom or disease requires the involvement of C3. In another embodiment, the present invention relates to a method for treating one or more retinal or ophthalmic diseases comprising administering a pharma- ceutical effective amount of an antibody or antigen-binding fragment to a patient in need thereof.

[0017] In one aspect, the invention provides use of the anti-C3 antibody or antigen-binding fragment thereof for the manufacture of a medicament for treating or preventing an ophthalmic or eye disease. In one aspect, the present invention relates to a method for treating retinopathy, proliferative retinopathy (PR) such as retinopathy of prematurity, diabetic retinopathy (DR) including ischemic retinopathy, proliferative diabetic retinopathy (PDR) and non-proliferative diabetic retinopathy, diabetic macular edema (DME), diabetic macular ischemia (DMI), age-related macular degeneration (AMD) including dry AMD and wet AMD, geographic atrophy (GA), retinitis pigmentosa, inherited retinal dystrophies, myopic degeneration, retinal vein occlusion, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, any retinal disease. Provided is an anti-C3 antibody or an antigen-binding fragment thereof for use in the treatment or prevention of a disease selected from the group consisting of secondary choroidal neovascular membrane, optic neuropathy, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal neovascularization, polypoidal choroidal vasculopathy, retinal vasculitis, retinal microaneurysm, retrolental fibroplasia, chorioretinitis, Fuchs' dystrophy, macular telangiectasia, Usher syndrome, paroxysmal nocturnal hemoglobinuria (PNH), and Stargardt's disease.

[0018] In another embodiment, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof for use in the treatment or prevention of a disease selected from the group consisting of age-related macular degeneration, geographic atrophy, neovascular glaucoma, and diabetic retinopathy. In a further preferred embodiment, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof for use in the treatment or prevention of geographic atrophy. In one aspect, the antibody of the present invention inhibits the activity of the classical complement pathway (CP), the lectin pathway (LP) and the alternative complement pathway (AP).Therefore, the present invention provides an anti-C3 antibody or an antigen-binding fragment thereof for use in a method for treating an ophthalmic or ocular disease by inhibiting the activity of the classical complement pathway (CP), the lectin pathway (LP) and the alternative complement pathway (AP).The present invention also provides an antigen-binding protein or a fragment thereof described above for use in a method for treating a complement C3-mediated disease or disorder by inhibiting the activity of choroid-localized complement C3. In one embodiment, the invention provides a method for diagnosing a disorder associated with complement C3 in a biological sample using an anti-C3 antibody or antigen-binding fragment thereof according to the invention. In one aspect, the invention provides a pharmaceutical composition comprising an anti-C3 antibody or antigen-binding fragment and a pharma- ceutically acceptable carrier.

[0019] In one embodiment, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising an anti-C3 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is administered by a parenteral, intravenous, intravitreal (IVT) or subcutaneous route of administration, preferably by the intravitreal route. In one embodiment, the invention provides a pharmaceutical composition comprising one or more polynucleotides encoding an antibody or antigen-binding fragment according to the invention and a pharma- ceutically acceptable carrier. In one embodiment, the invention provides an isolated polynucleotide or multiple polynucleotides encoding an antibody or antigen-binding fragment according to the invention. In one aspect, the present invention provides a method for producing a method for treating a cancer cell comprising: - a sequence encoding the heavy chain variable region according to SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 24, and / or - a sequence encoding the light chain variable region according to SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 The present invention provides an isolated polynucleotide or a plurality of polynucleotides comprising:

[0020] In one aspect, the invention provides an isolated polynucleotide comprising a sequence encoding an scFv as set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31. In one embodiment, the invention provides an isolated polynucleotide or an expression vector comprising a sequence encoding a heavy chain variable region as set forth in SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24, and / or a sequence encoding a light chain variable region as set forth in SEQ ID NO:21, SEQ ID NO:23 or SEQ ID NO:25. In one embodiment, the invention provides an expression vector comprising an isolated polynucleotide comprising a sequence encoding an scFv as set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31. In one embodiment, the invention provides an isolated polynucleotide or a viral vector comprising a sequence encoding a heavy chain variable region as set forth in SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, and / or a sequence encoding a light chain variable region as set forth in SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25. In one embodiment, the invention provides a viral vector comprising an isolated polynucleotide comprising a sequence encoding an scFv as set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31.

[0021] In one embodiment, the invention provides an expression vector or a host cell comprising an isolated polynucleotide or multiple polynucleotides comprising a sequence encoding a heavy chain variable region as set forth in SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24, and / or a sequence encoding a light chain variable region as set forth in SEQ ID NO:21, SEQ ID NO:23 or SEQ ID NO:25. In one embodiment, the invention provides an expression vector or a host cell comprising an isolated polynucleotide comprising a sequence encoding an scFv as set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31. In one embodiment, the invention provides a method for producing an anti-C3 antibody or an antigen-binding fragment thereof, comprising obtaining an expression vector or a host cell comprising an isolated polynucleotide or a plurality of polynucleotides comprising a sequence encoding a heavy chain variable region set forth in SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24, and / or a sequence encoding a light chain variable region set forth in SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:25; and culturing the host cell.

[0022] In one embodiment, the invention provides a method for producing an anti-C3 antibody or antigen-binding fragment thereof comprising obtaining an expression vector or a host cell comprising an isolated polynucleotide comprising a sequence encoding an scFv as set forth in SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31. In one embodiment, the method for producing an anti-C3 antibody or antigen-binding fragment thereof further comprises recovering and purifying the anti-C3 antibody or antigen-binding fragment thereof. [Brief description of the drawings]

[0023] [Figure 1] Figure 1 shows good retinal penetration after equimolar IVT injection with scFv in cynomolgus monkeys. Figure 1A shows retinal exposure levels of scFv intravitreal injection (40nmol scFv, brolucizumab, 1mg). Data source, brolucizumab BLA. Figure 1B shows retinal exposure levels of Fab intravitreal injection (40nmol Fab, ranibizumab, 2mg). Data source: Invest Ophthalmol Vis Sci. 2005 Feb;46(2):726-33. [Diagram 2] Figure 2 illustrates the binding of clone I to human complement C3. Figure 2A shows SEQ ID NO:55, a portion of the C3 sequence set forth in SEQ ID NO:47, with epitope residues in bold and underlined. Figure 2B shows the EM structure of the C3c:clone I complex, with the C3c structure shown in grey and the scFv structure in black ribbon representation. The final EM map is shown as a semi-transparent surface with a contour level of 0.214. [Diagram 3] Figure 1 shows the in vitro diffusion of Clone I and comparative compound A2 through porcine Bruch's membrane. Diffusion was measured in an Ussing chamber over a 72 hour period, and the amount of compound diffused through the Bruch's membrane is shown as the compound measured in the diffusion chamber compared to the total amount of compound (sample chamber and diffusion chamber). The total amount of compound was set to 100%. [Figure 4]Figure 1 shows the in vitro diffusion of Comparative Compound A3 and Comparative Compound A2 through porcine Bruch's membrane. Diffusion was measured in an Ussing chamber over 72 hours, and the amount of compound diffused through the Bruch's membrane is shown as the compound measured in the diffusion chamber compared to the total amount of compound (sample chamber and diffusion chamber). The total amount of compound was set to 100%. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] definition The general structure of an antibody or immunoglobulin is well known to those skilled in the art; these molecules are heterotetrameric glycoproteins, typically of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is covalently linked to a heavy chain by one disulfide bond to form a heterodimer, and a heterotrimeric molecule is formed through covalent disulfide bonds between the two identical heavy chains of the heterodimer. The light and heavy chains are linked together by one disulfide bond, while the number of disulfide bonds between the two heavy chains varies depending on the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains at its amino terminus a variable domain (V H = heavy chain variable region), followed by three or four constant domains (C H1 , C H2 , C H3 , and C H4 ), and C H1 and C. H2 Each light chain has two domains: an amino-terminal variable domain (V L = light chain variable region) and the carboxy-terminal constant domain (C L ) V L Domain is V H domain and is non-covalently associated with the C L The domains are usually linked together via disulfide bonds H1The light-chain variable domain is covalently linked to the heavy-chain variable domain. Certain amino acid residues are thought to form an interface between the light-chain variable domain and the heavy-chain variable domain (Chothia et al., 1985, J. Mol. Biol. 186:651-663).

[0025] Certain domains within the variable domains vary widely among different antibodies, i.e., are "hypervariable". These hypervariable domains contain residues that are directly involved in the binding and specificity of each particular antibody to its specific antigenic determinant. The hypervariability in both the light and heavy variable domains is concentrated in three segments known as complementarity determining regions (CDRs) or hypervariable loops (HVLs). The CDRs are described in Kabat et al., 1991, In: Sequences of Proteins of Immunological Interest, 5 th VLs are defined by comparison of sequences in the National Institutes of Health, Ed. Public Health Service, National Institutes of Health, Bethesda, Md., while HVLs are defined structurally according to the three-dimensional structure of the variable domains described by Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917. The precise amino acid sequence boundaries of a given CDR or FR can be further determined using additional nomenclature, such as the Aho numbering scheme described in Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun. 8; 309(3):657-70, or the "IMGT" numbering scheme described in Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003 Jan. 27(1):55-77.

[0026] The three CDRs in each of the heavy and light chains are separated by framework regions (FRs), which tend to have sequences that are less variable. From the amino terminus to the carboxy terminus of the heavy and light chain variable domains, the FRs and CDRs are arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The largely β-sheet conformation of the FRs brings the CDRs in each chain into close proximity with each other and with the CDRs of the other chain. The resulting conformation is involved in the antigen-binding site (see Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages 647-669), although not all CDR residues are directly involved in antigen binding. FR residues and Ig constant domains may contribute to antigen binding and / or mediate antibody effector functions. Some FR residues are believed to significantly affect antigen binding in at least three ways: by direct non-covalent binding to the epitope, by interacting with one or more CDR residues, and by influencing the interface between the heavy and light chains. The constant domains are not directly involved in antigen binding, but mediate various Ig effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

[0027] The light chains of vertebrate immunoglobulins are assigned to one of two clearly distinct classes, kappa (κ) and lambda (λ), based on the amino acid sequence of the constant domain. By comparison, the heavy chains of mammalian immunoglobulins are assigned to one of five major classes, IgA, IgD, IgE, IgG, and IgM, according to the sequence of the constant domain. IgG and IgA are further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, respectively. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the native immunoglobulin classes are well known. The terms "antibody," "anti-C3 antibody," "humanized anti-C3 antibody," and "variant humanized anti-C3 antibody" are used herein in the broadest sense and specifically encompass monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, such as variable domains and other antibody portions, that exhibit the desired biological activity, e.g., binding to C3.

[0028] The term "monoclonal antibody" (mAb) refers to an antibody from a population of substantially homogeneous antibodies; that is, the individual antibodies in the population are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant, "epitope." Thus, the modifier "monoclonal" indicates a substantially homogeneous population of antibodies directed against the same epitope, and is not to be construed as requiring production of the antibodies by any particular method. It should be understood that the monoclonal antibodies can be made by any technique or methodology known in the art, including, for example, the hybridoma method (Kohler et al., 1975, Nature 256:495), or recombinant DNA methods known in the art (see, e.g., U.S. Pat. No. 4,816,567), or methods for isolating recombinantly produced monoclonals using phage antibody libraries using the techniques described in Clackson et al., 1991, Nature 352: 624-628, and Marks et al., 1991, J. Mol. Biol. 222: 581-597.

[0029] A chimeric antibody consists of the heavy and light chain variable regions of an antibody from one species (e.g., a non-human mammal such as mouse) and the heavy and light chain constant regions of an antibody from another species (e.g., human), and can be obtained by linking a DNA sequence encoding the variable region of an antibody from a first species (e.g., mouse) to a DNA sequence of the constant region of an antibody from a second species (e.g., human) and transforming a host with an expression vector carrying the linked sequences, thereby enabling the host to produce the chimeric antibody. Alternatively, a chimeric antibody can also be an antibody in which one or more regions or domains of the heavy and / or light chain are identical to, homologous to, or variants of corresponding sequences in a monoclonal antibody from another immunoglobulin class or isotype, or from a consensus or germline sequence. Chimeric antibodies can include antibody fragments so long as such antibody fragments exhibit the desired biological activity of the parent antibody, e.g., binding to the same epitope (see, e.g., U.S. Pat. No. 4,816,567 and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855).

[0030] The terms "antibody fragment", "antigen-binding fragment", "anti-C3 antibody fragment", "humanized anti-C3 antibody fragment", "variant humanized anti-C3 antibody fragment" refer to a portion of a full-length anti-C3 antibody that retains the variable region or functional capability, such as specific C3 epitope binding. Examples of antibody fragments or antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, Fv, scFv and scFv-Fc fragments, diabodies, linear antibodies, single-chain antibodies, minibodies, diabodies formed from antibody fragments, single domain antibody (e.g., sdAb, sdFv, nanobodies, VHH) fragments and multispecific antibodies formed from antibody fragments. Full-length antibodies can be treated with enzymes such as papain or pepsin to generate useful antibody fragments. Papain digestion produces two identical antigen-binding antibody fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment. The Fab fragment also contains the constant domains of the light chain and the C domain of the heavy chain. H1 Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0031] The Fab' fragment is H1 They differ from Fab fragments by the presence of additional residues, including one or more cysteines, of the antibody hinge region at the C-terminus of the domain. F(ab')2 antibody fragments are pairs of Fab' fragments linked by cysteine ​​residues in the hinge region. Other chemical bonds of antibody fragments are also known. An "Fv" fragment contains a complete antigen recognition and binding site, consisting of a dimer of one heavy-chain variable domain and one light-chain variable domain in tight, non-covalent association. In this configuration, the three CDRs of each variable domain interact to form a V H -V L Collectively, the six CDRs confer antigen-binding specificity to the antibody, defining an antigen-binding site on the surface of the dimer. "Single-chain Fv" or "scFv" antibody fragments are fragments of the V H Domain and V L A single chain Fv variant is one that contains V domains, where these domains are present in a single polypeptide chain. Single chain Fvs can recognize and bind antigens. scFv polypeptides contain V domains that facilitate the formation of the desired three-dimensional structure for antigen binding by the scFv. H Domain and V LOptionally, a polypeptide linker may be located between the domains (see, for example, Pluckthun, 1994, In The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315). Such linkers include, but are not limited to, a repeating GGGGS amino acid sequence (SEQ ID NO: 43) or variants thereof. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Although scFvs do not generally contain antibody constant domain regions, the scFvs of the present invention may be linked or attached to antibody constant domain regions (e.g., antibody Fc domains) to alter various properties of the scFv, including, but not limited to, increased serum or tissue half-life. An scFv generally has a molecular weight of about 25 kDa and a hydrodynamic radius of about 2.5 nM.

[0032] In one embodiment, the antibodies and antibody fragments of the invention are single chain antibodies (scFv) or Fab fragments. In the case of scFv antibodies, the selected VL domain may be linked in either orientation to the selected VH domain by a flexible linker. Flexible linkers contemplated herein are peptide or polypeptide linkers of at least one amino acid in length. Preferably, the linker is 1-100 amino acids in length. More preferably, the linker is 5-50 amino acids in length, more preferably 10-40 amino acids in length, and even more preferably, the linker is 15-30 amino acids in length. Non-limiting examples of small molecule linkers that are often used include a sequence of glycine and serine amino acids called the GS mini linker. Preferred examples of linker sequences include (gly4ser)3 (SEQ ID NO: 45), (gly4ser)4 (SEQ ID NO: 46), (gly4ser) (SEQ ID NO: 44), (gly3ser) (SEQ ID NO: 56), gly3 and (gly3ser2)3 (SEQ ID NO: 65) (glyser2) (glyser1) (glyser2) (glyser3) (glyser4) (glyser5) (glyser6) (glyser7) (glyser8) (glyser9) (glyser10) (glyser11) (glyser12) (glyser13) (glyser14) (glyser15) (glyser16) (glyser17) (glyser18) (glyser19) (glyser20) (glyser21) (glyser22) (glyser33) (glyser34) (glyser35) (glyser36) (glyser37) (glyser38) (glyser39) (glyser39) (glyser39) (glyser39) (glyser39) (glyser3 x ser y )z Linkers such as Gly / Ser linkers of various lengths. The number of amino acids in these linkers can vary, for example, they can be 4 (e.g., GGGS) (SEQ ID NO: 56), 6 (e.g., GGSGGS) (SEQ ID NO: 57), 7 (e.g., GGGSGGS) (SEQ ID NO: 58) or multiples thereof, such as 2 or 3 or more repeats of these 4, 6 or 7 amino acids. In one embodiment, the antibody of the invention comprises a linker selected from the group consisting of the sequences set forth in SEQ ID NO: 43 (GGGGS), SEQ ID NO: 44 (GGGGSGGGGS), SEQ ID NO: 45 (GGGGSGGGGSGGGGS) and SEQ ID NO: 46 (GGGGSGGGGSGGGGSGGGGS). In a particular embodiment, the linker is SEQ ID NO: 46. The linker can also be a variant as set forth in Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448. Other linkers that can be used for the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. Immunother. 50:51-59. Further examples of linkers include the following: 7GS linker: SGGSGGGGS (SEQ ID NO:59); 8GS linkers: 9GS linker: GGGGSGGGS (SEQ ID NO:60); 18GS linker: GGGGSGGGGSGGGGGGGS (SEQ ID NO:61); 25GS linker: GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:62); 30GS linker: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:63); and 35GS linker: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO:64).

[0033] In the scFv according to the present disclosure, the arrangement of VL and VH may be either VL-linker-VH or VH-linker-VL, the former orientation being preferred. However, single VH or VL domain antibodies are also contemplated. In the case of Fab fragments, the selected light chain variable domain VL is fused to the constant region of the human Ig kappa chain, while the preferred heavy chain variable domain VH is fused to the first (N-terminal) constant domain CH1 of human IgG. Interchain disulfide bridges may be formed at the C-terminus of the constant domain or at other sites in the variable or constant domains. As used herein, a "VHH", "nanobody" or "heavy chain antibody" is an antigen-binding protein that contains a single heavy chain variable domain derived from species of the Camelidae family, including camels, llamas and alpacas. VHHs generally have a molecular weight of about 15 kDa. Other recognized antibody fragments include those that contain tandem Fd segments (V H -C H1 -V H -C H1 ) pairs. These "linear antibodies" can be bispecific or monospecific, e.g., as described in Zapata et al. 1995, Protein Eng. 8(10):1057-1062. A humanized antibody or humanized antibody fragment is a specific type of chimeric antibody, which includes immunoglobulin amino acid sequence variants or fragments thereof that can bind to a given antigen and that contain one or more FRs that have substantially the amino acid sequence of a human immunoglobulin and one or more CDRs that have substantially the amino acid sequence of a non-human immunoglobulin. This non-human amino acid sequence, often referred to as the "import" sequence, typically comes from an "import" antibody domain, particularly the variable domain. Usually, a humanized antibody contains at least the CDRs or HVLs of a non-human antibody inserted between the FRs of a human heavy or light chain variable domain.

[0034] The present invention describes certain humanized anti-C3 antibodies that contain CDRs derived from mouse, rabbit, llama or chimeric antibodies and inserted between the FRs of human germline sequence heavy and light chain variable domains. It is understood that certain non-human FR residues (e.g., from mouse, rabbit, llama or chimeric antibodies, etc.) may be important for the function of the humanized antibody, whereby certain human germline sequence heavy and light chain variable domain residues are altered to be identical to those of the corresponding non-human sequences. As used herein, the phrases "antibody of the invention" and "anti-C3 antibody of the invention" refer to an antibody or an antigen-binding fragment thereof directed against C3 as described herein. In one embodiment, said antibody of the invention is an scFv. In a specific embodiment, the antibody of the invention comprises a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises a CDR-H1 sequence of SEQ ID NO: 1, a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2 and 15, a CDR-H3 sequence of SEQ ID NO: 3; and wherein the VL comprises a CDR-L1 sequence of SEQ ID NO: 4, a CDR-L2 sequence selected from the group consisting of SEQ ID NO: 5 and 18, and a CDR-L3 sequence of SEQ ID NO: 6.

[0035] In one embodiment, the present invention relates to a humanized anti-C3 antibody or fragment thereof. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (e.g., contained in Fab, Fab', F(ab')2, Fabc, and Fv fragments) in which all or substantially all of the CDRs correspond to the CDRs of a non-human immunoglobulin, and specifically herein, the CDRs are of rabbit origin, and the FRs are consensus or germline sequence FRs of human immunoglobulins. In another aspect, the humanized anti-C3 antibody also comprises at least a portion of an immunoglobulin Fc region, typically the Fc region of a human immunoglobulin. Typically, the antibody has both a light chain and at least the variable domain of a heavy chain. The antibody also optionally comprises the CDRs of the heavy chain. H1 Area, hinge area, C H2 area, C H3 Area and / or C H4 It may include one or more of the regions. The humanized anti-C3 antibody may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. For example, the constant domain may be a complement fixing constant domain if it is desired that the humanized antibody exhibit cytotoxic activity, and the isotype is typically IgG1. If such cytotoxic activity is not desired, the constant domain may be of another isotype, such as IgG2, or a modified IgG1 sequence lacking cytotoxic activity, for example, containing the mutations N297A or L234A with L235A. Alternative humanized anti-C3 antibodies may contain sequences from more than one immunoglobulin class or isotype, and the selection of a particular constant domain to optimize the desired effector function is within the ordinary skill in the art. In a specific embodiment, the invention provides antibodies that are IgG1 antibodies, more particularly IgG1 antibodies characterized by reduced effector function.

[0036] The FR and CDR, or HVL, of the humanized anti-C3 antibody or its fragment, including scFv, need not correspond exactly to the parent sequence. For example, one or more residues in the imported CDR or HVL, or consensus or germline FR sequence, may be modified (e.g., mutagenized) by substitution, insertion, or deletion such that the resulting amino acid residue is no longer identical to the original residue at the corresponding position in any parent sequence, but the antibody still retains the binding function to C3. Such modifications are typically conservative rather than major. Usually, at least 75% of the humanized antibody residues, more frequently at least 90%, and most frequently more than 95%, or more than 98%, or more than 99%, correspond to the residues of the parent consensus or germline FR and imported CDR sequences.

[0037] The terms "consensus sequence" and "consensus antibody" refer to an amino acid sequence that contains the most frequently occurring amino acid residue at each position in all immunoglobulins of any particular class, isotype, or subunit structure, for example, in the human immunoglobulin variable domain. The consensus sequence may be based on immunoglobulins of a particular species or many species. It is understood that a "consensus" sequence, structure, or antibody encompasses the consensus human sequence as described in certain embodiments, and refers to an amino acid sequence that contains the most frequently occurring amino acid residue at each position in all human immunoglobulins of any particular class, isotype, or subunit structure. Thus, a consensus sequence has an amino acid sequence that has an amino acid at each position that is present in one or more known immunoglobulins, but may not be an exact duplicate of the entire amino acid sequence of any single immunoglobulin. The consensus sequence of the variable region is not derived from any naturally occurring antibody or immunoglobulin. Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. The FRs of the consensus sequences of the heavy and light chains, and variants thereof, provide sequences useful for preparing humanized anti-C3 antibodies. See, e.g., U.S. Patent Nos. 6,037,454 and 6,054,297.

[0038] Human germline sequences are naturally found in the human population. Human germline sequences correspond to antibody protein molecules encoded by variable (V), diversity (D) and joining (J) gene segments that rearrange to form recombinant genes. VDJ recombination results in the generation of a large but ultimately finite number of unmutated antibody proteins known as the germline repertoire. The combination of these germline genes generates antibody diversity. Germline antibody sequences for antibody light chains are derived from conserved human germline kappa or lambda v and j genes. Similarly, heavy chain sequences are derived from germline v, d and j genes (LeFranc, MP, and LeFranc, G, "The Immunoglobulin Facts Book" Academic Press, 2001). An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of the antibody's natural environment are contaminants that may interfere with diagnostic or therapeutic uses of the antibody, and may be enzymes, hormones, or other proteinaceous or nonproteinaceous solutes. In one aspect, the antibody is purified to at least greater than 95% isolation by weight of the antibody.

[0039] The term "antibody performance" refers to factors / properties that contribute to the antigen recognition of an antibody in vivo or the efficacy of an antibody. In a preferred embodiment, it refers to the ability of an antibody to prevent cytoskeleton breakdown in retinal cells. Changes in the amino acid sequence of an antibody can affect antibody properties such as folding and can also affect the initial antibody binding rate to the antigen (k a ), the dissociation constant of the antibody from the antigen (k d ), the affinity constant of the antibody for the antigen (K d The affinity of an antibody can be influenced by physical factors such as the affinity of the antibody, the conformation of the antibody, the stability of the protein, and the half-life of the antibody. As used herein, the term "affinity" refers to the strength of the interaction between the antigen-binding site of an antibody and the epitope to which it binds. As will be readily understood by one of skill in the art, antibody or antigen-binding protein affinity is measured by the dissociation constant (K DThe ability of an antigen-binding domain to bind to a particular antigenic determinant may be measured through enzyme-linked immunosorbent assays (ELISAs) or other techniques well known to those skilled in the art, such as surface plasmon resonance (SPR) techniques performed at 25° C. and analyzed on a BIAcore instrument (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0040] As used herein, the term "identical" or "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical or have a certain percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence. To determine percent identity, the sequences are aligned for optimal comparison (e.g., gaps can be introduced into the sequence of the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence contains an amino acid residue or nucleotide that is identical to the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (e.g., overlapping positions) x 100). In some embodiments, the two sequences being compared are of the same length (e.g., excluding additional sequences that extend beyond the sequences being compared), optionally after introducing gaps into the sequences as necessary. For example, when comparing variable region sequences, leader and / or constant domain sequences are not considered. In sequence comparison between two sequences, a "corresponding" CDR refers to a CDR that is at the same position in both sequences (e.g., CDR-H1 of each sequence).

[0041] The determination of percent identity or similarity between two sequences can be carried out using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotide searches can be carried out using the NBLAST program with score=100 and wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid encoding the protein of interest. To obtain amino acid sequences homologous to a protein of interest, BLAST protein searches can be performed using the XBLAST program with score=50 and wordlength=3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterative search that detects distant relationships between molecules (Id.). When utilizing the BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Another preferred, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art, including ADVANCE and ADAM, described in Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5, and FASTA, described in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search. If ktup=2, similar regions in the two sequences being compared are found by looking at aligned residue pairs, and if ktup=1, single aligned amino acids are examined. ktup can be set to 2 or 1 for protein sequences, or 1-6 for DNA sequences. If ktup is not specified, the default is 2 for proteins and 6 for DNA. Alternatively, protein sequence alignments may be performed using the CLUSTAL W algorithm as described by Higgins et al., 1996, Methods Enzymol. 266:383-402.

[0042] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include the progeny thereof. Thus, "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. The term "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sporting, or pet animals, such as dogs, horses, cats, cows, and the like. Preferably, the mammal is a human.

[0043] A "disease" or "disorder" as used herein is any condition that would benefit from treatment with a humanized anti-C3 antibody described herein, including chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disorder in question. The term "intravitreal injection" has its ordinary meaning in the art and refers to the introduction of an anti-C3 antibody or antigen-binding fragment thereof into the patient's vitreous. The term "subcutaneous administration" refers to the introduction of an anti-C3 antibody or antigen-binding fragment thereof under the skin of an animal or human patient by relatively slow, sustained delivery from a drug reservoir, preferably into a pocket between the skin and the underlying tissue. The pocket can be created by pinching or pulling the skin up and away from the underlying tissue.

[0044] The term "subcutaneous infusion" refers to the introduction of a drug under the skin of an animal or human patient, preferably into a pocket between the skin and the underlying tissue, by relatively slow, sustained delivery from a drug reservoir over a period of time, including but not limited to 30 minutes or less or 90 minutes or less. Optionally, the infusion may be performed by subcutaneous implantation of a drug delivery pump implanted under the skin of the animal or human patient, where the pump delivers a predetermined amount of drug over a predetermined period of time, for example 30 minutes, 90 minutes, or the length of a treatment regimen. The term "subcutaneous bolus" refers to drug administration beneath the skin of an animal or human patient, where the bolus drug delivery is less than about 15 minutes, in other embodiments less than 5 minutes, and in still other embodiments less than 60 seconds. In still other embodiments, administration occurs within a pocket between the skin and the underlying tissue, where the pocket can be created by pinching or pulling the skin up and away from the underlying tissue.

[0045] The term "therapeutically effective amount" is used to refer to an amount of anti-C3 antibody or antigen-binding fragment thereof that alleviates or improves one or more symptoms of the disorder being treated. In alleviating or improving, a therapeutically effective amount is an amount that results in a favorable outcome for the patient. Efficacy can be measured in a conventional manner depending on the condition being treated. For example, in ophthalmic or ocular diseases characterized by cells expressing C3, efficacy can be measured by determining the response rate, e.g., vision recovery, or by assessing the delay time to disease progression. The terms "treatment" and "therapy" and the like, as used herein, are meant to include therapeutic and prophylactic or suppressive measures for a disease or disorder that result in any clinically desirable or beneficial effect, including, but not limited to, alleviating or alleviating one or more symptoms, reversing, slowing or halting the progression of a disease or disorder. Thus, for example, the term treatment includes administering an anti-C3 antibody or an antigen-binding fragment thereof before or after the onset of symptoms of a disease or disorder, thereby preventing or eliminating one or more symptoms of a disease or disorder. As another example, the term includes administering an anti-C3 antibody or an antigen-binding fragment thereof after clinical signs of a disease to combat symptoms of a disease. Furthermore, administration of an anti-C3 antibody or an antigen-binding fragment thereof after onset and after the appearance of clinical symptoms includes "treatment" or "therapy" as used herein, if the administration affects clinical parameters of the disease or disorder, whether or not the treatment results in an improvement of the disease. Furthermore, so long as the compositions of the present invention, alone or in combination with another therapeutic agent, reduce or ameliorate at least one symptom of the disorder being treated compared to that symptom in the absence of use of an anti-C3 antibody composition or antigen-binding fragment thereof, the result should be considered to be an effective treatment of the underlying disorder, whether or not all symptoms of the disorder are reduced.

[0046] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products having information about the indications, methods of use, methods of administration, contraindications, and / or warnings concerning the use of such therapeutic products. Antibodies of the Invention In one embodiment, the present invention relates to an anti-C3 antibody or an antigen-binding fragment thereof. In a particular embodiment, the present invention provides a humanized anti-C3 antibody or an antigen-binding fragment thereof. In a further embodiment, the present invention provides a humanized monoclonal anti-C3 antibody or an antigen-binding fragment thereof.

[0047] In the initial characterization, a library of antibodies targeting C3, in particular scFvs targeting C3, was generated. We humanized and optimized these scFvs as illustrated in Example 4. We further proceeded to further engineer the CDRs and FRs with various modifications to improve affinity and optimize amino acids rarely present in human antibodies. Through various and exhaustive optimization steps, we developed highly promising therapeutic molecules, in particular humanized scFvs directed against C3 with enhanced properties as disclosed herein.

[0048] The sequences of the CDRs of the antibodies of the invention are shown below in Table 1. Table 1 shows the CDRs according to the Kabat nomenclature. [Table 1] In one embodiment, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) and a variable light chain (VL): - the VH comprises the CDR-H1 sequence of SEQ ID NO: 1, a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2 and 15, and a CDR-H3 sequence of SEQ ID NO: 3; and - the VL comprises a CDR-L1 sequence of SEQ ID NO: 4, a CDR-L2 sequence selected from the group consisting of SEQ ID NO: 5 and 18, and a CDR-L3 sequence of SEQ ID NO: 6; An anti-C3 antibody or an antigen-binding fragment thereof is provided.

[0049] In a particular embodiment, the present invention relates to an anti-C3 antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) and a variable light chain (VL), wherein the VH is selected from the group consisting of a CDR-H1 sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 1, a CDR-H2 sequence of SEQ ID NO: 2 or 15 or an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 2 or 15, and / or a CDR-H3 sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 80%, at least 90%, or at least 95% identical to the amino acid sequence of SEQ ID NO: 3. and / or the VL comprises a CDR-L1 sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 80%, at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 4, a CDR-L2 sequence of SEQ ID NO: 5 or 18 or an amino acid sequence that is at least 80%, at least 90% or at least 95% identical to the amino acid sequence of SEQ ID NO: 5 or 18, and / or a CDR-L3 sequence of SEQ ID NO: 6 or an amino acid sequence that is at least 80% or at least 85% identical to the amino acid sequence of SEQ ID NO: 6.

[0050] In certain embodiments, the invention provides an anti-C3 antibody, or antigen-binding fragment thereof, comprising: - the VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 2 and the CDR-H3 sequence of SEQ ID NO: 3; and the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6, or - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 15 and the CDR-H3 sequence of SEQ ID NO: 3; and VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6; - the VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 15 and the CDR-H3 sequence of SEQ ID NO: 3; and the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 18 and the CDR-L3 sequence of SEQ ID NO: 6, or - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 2 and the CDR-H3 sequence of SEQ ID NO: 3; and VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 18 and the CDR-L3 sequence of SEQ ID NO: 6. The present invention provides an anti-C3 antibody or an antigen-binding fragment thereof.

[0051] In another embodiment, the present invention provides - a heavy chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 20, 22 or 24; and / or - a light chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 21, 23 or 25 The present invention provides an anti-C3 antibody or an antigen-binding fragment thereof comprising the following: -

[0052] In yet another embodiment, the present invention provides a method for producing - a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 20, 22 or 24; and - a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 21, 23 or 25 An anti-C3 antibody or an antigen-binding fragment thereof comprising: - the VH comprises a CDR-H1 sequence of SEQ ID NO: 1, a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2 and 15, and a CDR-H3 sequence of SEQ ID NO: 3; and - the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence selected from the group consisting of SEQ ID NO: 5 and 18, and the CDR-L3 sequence of SEQ ID NO: 6; An anti-C3 antibody or an antigen-binding fragment thereof is provided. In another embodiment, the present invention provides - a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 20; and - a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 21 An anti-C3 antibody or an antigen-binding fragment thereof comprising: - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 2 and the CDR-H3 sequence of SEQ ID NO: 3; and VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6; An anti-C3 antibody or an antigen-binding fragment thereof is provided.

[0053] In another embodiment, the present invention provides - a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 22; and - a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 23 An anti-C3 antibody or an antigen-binding fragment thereof comprising: - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 15 and the CDR-H3 sequence of SEQ ID NO: 3; and VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6; An anti-C3 antibody or an antigen-binding fragment thereof is provided. In another embodiment, the present invention provides - a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 24; and - a light chain variable region comprising an amino acid sequence that is at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 25 An anti-C3 antibody or an antigen-binding fragment thereof comprising: - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 15 and the CDR-H3 sequence of SEQ ID NO: 3; and VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 18 and the CDR-L3 sequence of SEQ ID NO: 6; An anti-C3 antibody or an antigen-binding fragment thereof is provided.

[0054] In one embodiment, the invention provides an anti-C3 antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20, 22 or 24; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21, 23 or 25. In another embodiment, the present invention provides a. a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO:20 and SEQ ID NO:21, respectively; b. a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO:22 and SEQ ID NO:23, respectively; or c. A variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:25, respectively. The present invention provides an anti-C3 antibody or an antigen-binding fragment thereof, comprising:

[0055] In yet another embodiment, the invention provides an anti-C3 antibody, or antigen-binding fragment thereof, comprising: a. the antibody is designated "clone I", a variable heavy chain comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 20 and a variable light chain comprising, preferably consisting of, the amino acid sequence of SEQ ID NO: 21; b. a variable heavy chain comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO: 22 and a variable light chain comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO: 23, the antibody being designated "clone II"; c. The antibody is designated "Clone III", a variable heavy chain comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:24 and a variable light chain comprising, and preferably consisting of, the amino acid sequence of SEQ ID NO:25. The present invention provides an anti-C3 antibody or an antigen-binding fragment thereof comprising the following:

[0056] The following table sets forth the CDRs of antibodies according to the invention by various known nomenclature systems, e.g., by Kabat; by CCG (Chemical Computing Group, exemplified in Almagro et al., Proteins 2011; 79:3050-3066 and Maier et al, Proteins 2014; 82:1599-1610); by Chothia; and / or by Aho. Table 2 further summarizes the amino acid sequences of the CDRs of the antibodies according to the invention. [Table 2] TIFF2024546183000003.tif229149

[0057] Thus, in a particular aspect, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) and a variable light chain (VL): - the VH comprises a CDR-H1 sequence selected from the group consisting of SEQ ID NOs: 1, 7, 8 and 10, a CDR-H2 sequence selected from the group consisting of SEQ ID NOs: 2, 9, 11, 15, 16 and 17, a CDR-H3 sequence selected from the group consisting of SEQ ID NOs: 3 and 12; and - the VL comprises a CDR-L1 sequence selected from the group consisting of SEQ ID NOs: 4 and 13, a CDR-L2 sequence selected from the group consisting of SEQ ID NOs: 5, 14, 18 and 19, and a CDR-L3 sequence of SEQ ID NO: 6; The present invention relates to an anti-C3 antibody or an antigen-binding fragment thereof.

[0058] In a specific aspect, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) and a variable light chain (VL): - the VH comprises the CDR-H1 sequence of SEQ ID NO: 7, the CDR-H2 sequence of SEQ ID NO: 2 and the CDR-H3 sequence of SEQ ID NO: 3; and the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6, or - the VH comprises the CDR-H1 sequence of SEQ ID NO: 8, the CDR-H2 sequence of SEQ ID NO: 9 and the CDR-H3 sequence of SEQ ID NO: 3; and the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6, or - the VH comprises the CDR-H1 sequence of SEQ ID NO: 10, the CDR-H2 sequence of SEQ ID NO: 11 and the CDR-H3 sequence of SEQ ID NO: 12; and the VL comprises the CDR-L1 sequence of SEQ ID NO: 13, the CDR-L2 sequence of SEQ ID NO: 14 and the CDR-L3 sequence of SEQ ID NO: 6, or - the VH comprises the CDR-H1 sequence of SEQ ID NO: 7, the CDR-H2 sequence of SEQ ID NO: 15 and the CDR-H3 sequence of SEQ ID NO: 3; and the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6, or - the VH comprises the CDR-H1 sequence of SEQ ID NO: 8, the CDR-H2 sequence of SEQ ID NO: 16 and the CDR-H3 sequence of SEQ ID NO: 3; and the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6, or - the VH comprises the CDR-H1 sequence of SEQ ID NO: 10, the CDR-H2 sequence of SEQ ID NO: 17 and the CDR-H3 sequence of SEQ ID NO: 12; and the VL comprises the CDR-L1 sequence of SEQ ID NO: 13, the CDR-L2 sequence of SEQ ID NO: 14 and the CDR-L3 sequence of SEQ ID NO: 6, or - the VH comprises the CDR-H1 sequence of SEQ ID NO: 7, the CDR-H2 sequence of SEQ ID NO: 15 and the CDR-H3 sequence of SEQ ID NO: 3; and the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 18 and the CDR-L3 sequence of SEQ ID NO: 6, or - the VH comprises the CDR-H1 sequence of SEQ ID NO: 8, the CDR-H2 sequence of SEQ ID NO: 16 and the CDR-H3 sequence of SEQ ID NO: 3; and the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 18 and the CDR-L3 sequence of SEQ ID NO: 6, or - VH comprises the CDR-H1 sequence of SEQ ID NO: 10, the CDR-H2 sequence of SEQ ID NO: 17 and the CDR-H3 sequence of SEQ ID NO: 12; and VL comprises the CDR-L1 sequence of SEQ ID NO: 13, the CDR-L2 sequence of SEQ ID NO: 19 and the CDR-L3 sequence of SEQ ID NO: 6; The present invention relates to an anti-C3 antibody or an antigen-binding fragment thereof.

[0059] Exemplary variable heavy and variable light chain sequences according to the invention are shown in Table 3. [Table 3]

[0060] The sequences of exemplary scFvs of the invention are shown in Table 4. [Table 4]

[0061] In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof of the present invention is selected from the group consisting of single chain variable fragments (scFv), Fab fragments, Fab' fragments, Fv fragments, diabodies, small molecule antibody mimetics. In an alternative embodiment, the anti-C3 antibody or antigen-binding fragment thereof is selected from the group consisting of single domain antibodies, e.g., sdAb, sdFv, nanobodies, V-Nar, and VHH. In this particular embodiment, the anti-C3 antibody or antigen-binding fragment thereof comprises a variable heavy chain. In a further embodiment, the variable heavy chain comprises a CDR-H1 sequence of SEQ ID NO: 1, a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2 and 15, and a CDR-H3 sequence of SEQ ID NO: 3. In a specific embodiment, the variable heavy chain comprises the amino acid sequence of SEQ ID NO: 20, 22, or 24. In one embodiment, the antibody of the invention is a single chain fragment that comprises a linker, preferably comprising the linker set forth in SEQ ID NO:46 (GGGGSGGGGSGGGGSGGGGS). In one embodiment, the antibody of the invention is a single chain fragment comprising one sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30 and 31.

[0062] In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention can inhibit pathways of complement activation, including the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP). In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention is capable of binding to complements C3 and C3b. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention may prevent the formation of C3 convertase. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention is capable of penetrating Bruch's membrane. In one embodiment, an anti-C3 antibody or antigen-binding fragment thereof according to the invention has approximately equal binding affinity for C3 and C3b.

[0063] In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention has an activity against C3a, iC3b, C4, C4b, C5 and / or C5b of about 10 -4 It has a binding affinity equal to or less than M (i.e., weak). In one embodiment, an anti-C3 antibody or antigen-binding fragment thereof according to the invention has weaker binding affinity for C3a, iC3b, C4, C4b, C5 and / or C5b compared to its binding affinity for C3 and C3b. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention has no binding affinity for C3a, iC3b, C4, C4b, C5 and / or C5b.

[0064] In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention can inhibit the C3 convertase amplification loop. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention can inhibit choroidal C3 activity. The anti-C3 antibodies of the invention, or antigen-binding fragments thereof according to the invention, bind to human C3 with high affinity. In one embodiment related to this aspect, the anti-C3 antibodies of the invention bind to human C3 with a K D <50 nM, preferably K D <15 nM, preferably K D <10 nM, preferably K D <7 nM, preferably K D <1 nM, preferably K D <0.5 nM, preferably K D <0.2 nM, preferably K D <0.15 nM, preferably K D <0.10 nM, preferably K D <0.05 nM, preferably K D <0.04 nM, more preferably K D In one embodiment, the anti-C3 antibody of the present invention binds to human C3 with a K between 0.16 and 0.023 nM, as exemplified in Example 8. D Combine with.

[0065] In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention binds to human C3b with a K D <50 nM, preferably K D <15 nM, preferably K D <10 nM, preferably K D <7 nM, preferably K D <1 nM, preferably K D <0.5 nM, preferably K D <0.2 nM, preferably K D <0.15 nM, preferably K D <0.10 nM, more preferably K D <0.05 nM. In a further embodiment, the anti-C3 antibody of the invention binds to human C3 with a K comprised between 0.15 nM and 0.05 nM, as exemplified in Example 8. D Combine with. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention has an activity against C3 and C3b of about 10 -8 M ~ about 10 -14 In certain embodiments, the antibodies according to the invention have a binding affinity of about 10 for C3 and C3b. -10 M ~ about 10 -12 In certain embodiments, the anti-C3 antibodies of the invention have a binding affinity of at least about 10 to C3 and C3b. -8 M (stronger), at least about 10 -9 M (stronger), at least about 10 -10 M (stronger), at least about 10 -11 M (stronger) or at least about 10 -12 In one embodiment, the antibodies according to the invention have approximately equal binding affinities to C3 and C3b. For example, but not limited to, the antibodies according to the invention have approximately 10 binding affinities to C3. -10 M binding affinity for C3b, and approximately 10 -10 In certain embodiments, the antibodies according to the invention may have a binding affinity for C3 of about 10 -11 M binding affinity for C3b, and approximately 10 -11 In another embodiment, the antibody according to the invention has a binding affinity for C3 of about 10 -12 M binding affinity for C3b, and approximately 10 -12 It has a binding affinity of M.

[0066] In an alternative embodiment, the binding affinity for C3 is within 10-fold of the binding affinity for C3b. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the invention exhibits cross-reactivity with cynomolgus monkey C3. The inventors have shown in Example 8 that the antibodies of the invention also bind to cynomolgus monkey C3. Cynomolgus monkey (Macaca fascicularis) C3 is 95.1% identical to human C3, and the cross-reactivity allows for preclinical and toxicology testing of the anti-C3 antibodies of the invention in relevant animal models. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention has binding affinity for C3 disease-associated single nucleotide polymorphisms (SNPs), more precisely C3 SNP P314L and C3 SNP R102G. The present inventors have also shown in Example 8 that the antibodies of the present invention bind to disease-associated SNPs of human C3, and that the antibodies of the present invention can treat a wide range of patients suffering from ophthalmic or ocular diseases. The high binding affinity of the anti-C3 antibody or its antigen-binding fragment according to the present invention contributes to the extension of the neutralization time of C3 after intravitreal injection, and further allows for a reduction in injection frequency. Furthermore, the high binding affinity further allows for administration at a lower dose, limiting the possibility of side effects. The favorable binding affinity and the reduction in injection frequency significantly improve the efficacy of treatment for patients in need thereof.

[0067] Due to their high binding affinity, their favorable half-life and their ability to be highly concentrated, the anti-C3 antibodies of the present invention allow for extended intervals between doses, also providing valuable benefits to patients, particularly improved drug observance and compliance. The anti-C3 antibody or antigen-binding fragment thereof according to the present invention may have a therapeutically effective duration longer than one month, which may be a long duration compared to other therapeutic agents. The extended therapeutically effective duration may be due to the molar concentration of the anti-C3 antibody or antigen-binding fragment thereof according to the present invention, which may reach a high concentration of 7 mM. The anti-C3 antibody or antigen-binding fragment thereof according to the present invention can be more easily injected into the eye compared to other therapeutic agents. In one embodiment, the anti-C3 antibody of the present invention does not contain PEG, and therefore has a reduced viscosity, as illustrated in Example 13. Therefore, the viscosity of the anti-C3 antibody of the present invention is expected to be lower than that of other therapeutic agents. A solution with a low viscosity, such as a solution with a viscosity of 20 centipoise (cP) or less, is more easily injected into the eye due to reduced back pressure. The present inventors have shown that the anti-C3 antibody or antigen-binding fragment thereof according to the present invention exhibits excellent pharmaceutical properties, as exemplified by improved stability. In Example 11, the present inventors have demonstrated that the anti-C3 antibody or antigen-binding fragment thereof according to the present invention has improved thermal stability. These results illustrate that the antibody of the present invention maintains its native and active conformation at physiological temperatures. The higher thermal transition midpoint (T m It is noteworthy that the T reflects improved stability of the protein at low temperatures. The inventors have thereby demonstrated that the anti-C3 antibody or antigen-binding fragment thereof according to the present invention exhibits improved thermostability properties, contributing to improved therapeutic efficacy while allowing for a reduction in the dose and frequency of injections to patients. In addition, T m demonstrates improved shelf life and stability of therapeutic products.

[0068] In a further aspect, the anti-C3 antibody or antigen-binding fragment thereof according to the invention has been demonstrated to have a low immunogenicity risk as described in Examples 4 and 12. These results confirm that the antibodies of the invention are highly suitable and promising for the treatment of ophthalmic or ocular diseases, since they exhibit a low immunogenicity risk and / or a low risk of generating anti-drug antibodies (ADA). The complement system is an important part of the innate immune system that is involved in tissue injury in many inflammatory and autoimmune diseases. The complement system comprises over 30 cell-associated and circulating proteins (e.g., C1, C1q, C1r, C1s, C2, C3, C3a, C3b, C4, factor B, factor D, factor H, factor I). There are three major pathways that activate complement: the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP).

[0069] The three complement pathways are initiated by different factors and each results in cleavage of complement component C3. The classical pathway is usually triggered by the interaction of an antigen with a specific antibody of the correct isotype or subclass; immunoglobulin M (IgM), IgG3, and IgG1 are the most efficient activators of the classical pathway. It induces a conformational change in the C1 complex, allowing the cleavage of C4 and C2 generating the C4bC2b complex. C4bC2b acts as the C3 convertase of the classical pathway. The lectin complement pathway is triggered by the binding of a series of related proteins called C-type lectins, mannan-binding lectins (MBL; also known as mannose-binding lectins) or ficolins (L-ficolin, H-ficolin and M-ficolin) to terminal sugars expressed on glycoproteins or envelope polysaccharides found on the surfaces of microorganisms.

[0070] The alternative pathway is initiated by the slow hydrolysis of circulating C3, exposing an internal thioester group, a phenomenon termed "C3 tickover." Binding of the alternative pathway-specific proteins factor B, factor D, and properdin to the hydrolyzed C3, or to the complement cleavage fragment C3b, results in further activation of C3. C3 cleaved to the form of C3b can then interact with polysaccharides or proteins on the surface of microorganisms, or with endotoxins (bacterial lipopolysaccharides), initiating alternative pathway activation and generating MAC in a manner similar to that which occurs during classical pathway activation. In certain embodiments, the anti-C3 antibodies or antigen-binding fragments thereof according to the invention are selected for their ability to inhibit one or more of the complement pathways, the classical pathway, the alternative pathway, and the lectin pathway. In certain embodiments, the anti-C3 antibodies of the invention are selected for their ability to inhibit all three complement pathways, the classical pathway, the alternative pathway, and the lectin pathway. In certain embodiments, the anti-C3 antibodies or antigen-binding fragments thereof according to the invention can inhibit all three complement pathways in the eye.

[0071] Typically, the functional potency of the anti-C3 antibody or antigen-binding fragment thereof according to the present invention in inhibiting the classical, alternative and lectin pathways can be determined by a complement inhibition assay or a hemolytic assay as described in Example 7. The potency can be expressed as IC50 (half maximal inhibitory concentration). Thus, in one embodiment, the antibody or fragment thereof according to the present invention: - Inhibits the classical pathway (CP) with a potency between 70-80nM as measured by the complement inhibition assay; - inhibits the lectin pathway (LP) with a potency comprised between 340 and 360 nM as measured by the complement inhibition assay; and - Inhibits the alternative pathway (AP) with a potency comprised between 60-70nM as measured by complement inhibition assay. The ability of the anti-C3 antibodies or antigen-binding fragments thereof according to the invention to inhibit all three complement pathways further improves their therapeutic potential in the treatment of ophthalmic or ocular diseases. In one embodiment, the ability of the antibodies of the invention to inhibit all three complement pathways further improves their therapeutic potential in the treatment of ophthalmic or ocular diseases, particularly AMD or GA. Without being bound by theory, inhibiting all three complement pathways may improve the therapeutic potential of the anti-C3 antibodies of the invention by preventing the disease-promoting effects of one active pathway from compensating for the inactivated pathways of the others.

[0072] Thus, in certain embodiments, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention can inhibit all three complement pathways in the choroidal region of the eye. The choroidal region is a layer containing blood vessels that covers the back of the eye and is located between the retina and the sclera. The choroidal region is divided into four layers: Hurler's layer, Sattler's layer, choriocapillaris, and Bruch's membrane. Bruch's membrane, also known as the vitreous lamina, is the innermost layer of the choroid and is adjacent to the retinal pigment epithelium (RPE). In certain embodiments, the anti-C3 antibody of the present invention can penetrate or diffuse across Bruch's membrane and enter other layers of the choroid, including but not limited to the choriocapillaris. The retina has a substantial physical barrier that can prevent large molecules, such as full-length immunoglobulins, from penetrating deeper layers, which can result in reduced therapeutic efficacy (Jackson et al. Invest Ophthalmol Vis Sci. 2003;44(5):2141-6). In contrast, smaller antibody derivatives can penetrate deeper into the retina. Exemplary antibody derivatives having a molecular weight of about 60 kDa or less are antibody fragments, including, but not limited to, Fab, Fab' fragments, scFab, scFv, Fv fragments, nanobodies, VHH, dAb, V-Nar, sdAb, sdFv, and bispecific and bivalent antibodies, such as single chain diabodies (scDbs) or DARTs. In certain embodiments, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention has a molecular weight of about 60 kDa or less, e.g., about 55 kDa, about 50 kDa, about 45 kDa, about 40 kDa, about 35 kDa, about 30 kDa, about 25 kDa, about 20 kDa, about 15 kDa or less. In one embodiment, the scFv format allows for a dosing frequency of 3 months, which represents a significant improvement over existing therapeutic approaches for treating ophthalmic or ocular diseases that require more frequent administration. Thus, the scFvs of the present invention significantly improve patient compliance.

[0073] The present inventors have shown in Example 10 that the anti-C3 antibody or antigen-binding fragment thereof according to the present invention (clone I) exhibits a high diffusion capacity through Bruch's membrane, especially when compared to comparative compound A2 and comparative compound A3, as described in Example 2. Thus, in one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention can penetrate or diffuse across Bruch's membrane, in part due to their size being low enough to facilitate penetration. In certain embodiments, the size of the antibody of the present invention is measured by molecular weight. In certain embodiments, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention has a molecular weight that is less than about 60 kDa. In certain embodiments, the anti-C3 antibody of the present invention is about 20 kDa to about 30 kDa or about 10 kDa to about 20 kDa. In certain embodiments, the anti-C3 antibody of the present invention is about 25 kDa. In certain embodiments, the anti-C3 antibody of the present invention is about 15 kDa. In certain embodiments, the size of the anti-C3 antibodies or antigen-binding fragments thereof according to the present invention is measured by their hydrodynamic radius. In certain embodiments, the anti-C3 antibodies of the present invention have a hydrodynamic radius of about 3.0 nm or less. In certain embodiments, the anti-C3 antibodies of the present invention have a hydrodynamic radius of about 2.5 nm or less. In certain embodiments, the anti-C3 antibodies or antigen-binding fragments thereof according to the present invention have a hydrodynamic radius of about 2.0 nm or less.

[0074] Humanized and Amino Acid Sequence Variants Furthermore, variant anti-C3 antibodies and antibody fragments may be engineered based on the set of CDRs identified in the sequences shown in SEQ ID NOs: 1-6, 15 and 18. It is understood that in some embodiments, in said variants of anti-C3 antibodies and antibody fragments, the amino acid sequences of the CDRs remain unchanged, but the surrounding regions, e.g., FR regions, may be engineered. Amino acid sequence variants of anti-C3 antibodies or fragments thereof may be prepared by introducing appropriate nucleotide changes into the anti-C3 antibody DNA or by peptide synthesis. Such variants include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the anti-C3 antibodies or fragments thereof of the examples herein. Any combination of deletions, insertions and substitutions may be made to arrive at the final construct, so long as the final construct possesses the desired characteristics. Amino acid changes may also alter post-translational processes of the humanized or variant anti-C3 antibodies or antigen-binding fragments thereof, such as by altering the number or position of glycosylation sites.

[0075] Another type of amino acid variant of an antibody involves altering the original glycosylation pattern of the antibody, the term "altering" in this context meaning deleting one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites that were not previously present in the antibody. In some aspects, the present invention includes nucleic acid molecules encoding amino acid sequence variants of the anti-C3 antibody or antigen-binding fragment thereof described herein. Nucleic acid molecules encoding amino acid sequence variants of the anti-C3 antibody or antigen-binding fragment thereof are prepared by various methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation of pre-prepared variant or non-variant versions of the anti-C3 antibody or antigen-binding fragment thereof by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis.

[0076] In certain embodiments, the anti-C3 antibody of the present invention is an antibody fragment. There are techniques developed for the production of antibody fragments. Fragments can be derived through proteolytic digestion of intact antibodies (see, for example, Morimoto et al., 1992, Journal of Biochemical and Biophysical Methods 24:107-117, and Brennan et al., 1985, Science 229:81). Alternatively, fragments can be produced directly in recombinant host cells. For example, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (see, for example, Carter et al., 1992, Bio / Technology 10:163-167). By another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to those skilled in the art. Anti-C3 antibodies and their antigen-binding fragments may include modifications. Variants of the antibodies provided herein may be generated by introducing deletions, substitutions, additions and / or modifications into the framework and / or into the CDRs. The antibody variants may then be tested for the desired function using the methods described herein. Any combination of deletions, substitutions, additions, modifications and insertions may be made into the antigen-binding protein or fragment thereof, as long as the generated variants have the desired characteristics that can be screened using appropriate methods.

[0077] As used herein, "conservative substitution" refers to a modification that maintains the functional properties of a parent antibody. For example, conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having similar properties. For example, alanine (A) is replaced with valine (V); arginine (R) is replaced with lysine (K); asparagine (N) is replaced with glutamine (Q); aspartic acid (D) is replaced with glutamic acid (E); cysteine ​​(C) is replaced with serine (S); glutamic acid (E) is replaced with aspartic acid (D); glycine (G) is replaced with alanine (A); histidine (H) is replaced with arginine (R) or lysine (K). replacing isoleucine (I) with leucine (L); replacing methionine (M) with leucine (L); replacing phenylalanine (F) with tyrosine (Y); replacing serine (S) with threonine (T); replacing tryptophan (W) with tyrosine (Y); replacing phenylalanine (F) with tryptophan (W); and / or replacing valine (V) with leucine (L) and vice versa.

[0078] In certain embodiments, it may be desirable to use anti-C3 antibody fragments rather than intact antibodies. It may be desirable to modify the antibody fragment to increase its serum half-life. This can be done, for example, by incorporating a salvage receptor binding epitope into the antibody fragment. In one method, the appropriate region of the antibody fragment can be modified (e.g., mutated), or the epitope can be incorporated into a peptide tag, which can then be fused to either end or the middle of the antibody fragment, for example, by DNA synthesis or peptide synthesis. See, for example, WO 96 / 32478. In other embodiments, the invention includes covalent modifications of anti-C3 antibodies or antigen-binding fragments. Covalent modifications include modifications of cysteinyl, histidyl, lysinyl and amino-terminal residues, arginyl, tyrosyl, carboxyl side groups (aspartyl or glutamyl), glutaminyl and asparaginyl, or seryl, or threonyl residues. Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. Such modifications can be made by chemical synthesis or, where appropriate, by enzymatic or chemical cleavage of the antibody or fragment thereof. Other types of covalent modifications of antibodies can be introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment thereof with organic derivatizing agents capable of reacting with selected side chains or amino- or carboxy-terminal residues.

[0079] Removal of any carbohydrate moieties present on the antibody or fragment thereof can be accomplished chemically or enzymatically. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52, and Edge et al., 1981, Anal. Biochem., 118:131. Enzymatic cleavage of carbohydrate moieties on the antibody can be accomplished by the use of a variety of endo- and exoglycosidases as described by Thotakura et al., 1987, Meth. Enzymol 138:350. Another type of useful covalent modification involves linking the antibody to one of a variety of nonproteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in one or more of U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. The variant antibodies or antigen-binding fragments thereof described herein, such as variants comprising sequences with a certain level of percent identity to the sequences disclosed herein, preferably retain the functional capabilities of the antibodies or antigen-binding fragments thereof described above, such as specific C3 epitope binding that can inhibit pathways of complement activation, including the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP), can bind complement C3 and C3b, can prevent the formation of C3 convertase, and / or can penetrate Bruch's membrane.

[0080] Epitope Binding In one aspect, the invention provides antibodies or antigen-binding fragments thereof that recognize a specific "C3 epitope." As used herein, the term "C3 epitope" refers to a molecule (e.g., a peptide) or a fragment of a molecule that can bind to an anti-C3 antibody or antigen-binding fragment thereof. These terms further include, for example, a C3 antigenic determinant recognized by any of the antibodies or antibody fragments of the invention. A C3 antigen epitope may be comprised in a protein, a protein fragment, a peptide, or the like. Epitopes are most commonly proteins, short oligopeptides, oligopeptide mimetics (i.e., organic compounds that mimic the antibody binding properties of the C3 antigen), or combinations thereof. In the context of epitope binding, the phrase "binding within amino acid region X through Y..." means that an anti-C3 antibody or antigen-binding fragment thereof according to the invention binds to at least one amino acid residue within the amino acid region specified in the sequence.

[0081] The antibody or antigen-binding fragment of the invention binds to an epitope of human complement C3 set forth in SEQ ID NO: 47. In one embodiment, the antibody of the invention binds to an epitope of the D chain of human complement C3c set forth in SEQ ID NO: 48. SEQ ID NO: 48 is available online at PDB: 2A74_D. The antibody or antigen-binding fragment of the invention binds to an epitope of human complement C3 set forth in SEQ ID NO: 47. In another embodiment, the antibody of the invention binds to an epitope of the D chain of human complement C3c set forth in SEQ ID NO: 48. SEQ ID NO: 48 is available online at PDB: 2A74_D. It has been found that the anti-C3 antibody or antigen-binding fragment thereof according to the present invention binds to a unique epitope of human complement C3. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof binds to at least one amino acid residue within amino acid 366 to amino acid 478 of human complement C3 as set forth in SEQ ID NO:47.

[0082] In one embodiment, the present invention provides an anti-C3 antibody or an antigen-binding fragment thereof that binds to at least one amino acid residue selected from the group consisting of residues 366, 392 to 396, 413 to 421, 425, 427, 442, 453, and 478 of human complement C3 set forth in SEQ ID NO: 47. In one embodiment, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof that binds to all of residues 366, 392 to 396, 413 to 421, 425, 427, 442, 453, and 478 of human complement C3 set forth in SEQ ID NO: 47. In one embodiment, the present invention relates to an anti-C3 antibody or antigen-binding fragment thereof that binds to at least one amino acid residue within amino acid residues 344 to 456 of the D chain of human complement C3c set forth in SEQ ID NO: 48. In one embodiment, the present invention relates to an anti-C3 antibody or an antigen-binding fragment thereof that binds to at least one residue selected from the group consisting of residues 344, 370 to 374, 391 to 399, 403, 405, 420, 431, and 456 of the D chain of human complement C3c set forth in SEQ ID NO:48.

[0083] In one embodiment, the antibody or antigen-binding fragment thereof of the present invention binds to all of residues 344, 370 to 374, 391 to 399, 403, 405, 420, 431, and 456 of the D chain of human complement C3c set forth in SEQ ID NO:48. In one embodiment, the present invention relates to an anti-C3 antibody or an antigen-binding fragment thereof that binds to at least one amino acid residue within amino acid residue 369 to amino acid residue 418 of the D chain of human complement C3c set forth in SEQ ID NO:48. In another embodiment, the present invention relates to an anti-C3 antibody or an antigen-binding fragment thereof that binds to at least one residue selected from the group consisting of residues 369 to 379, 389, 391 to 401, 403, and 418 of the D chain of human complement C3c set forth in SEQ ID NO:48. In another embodiment, the antibody of the present invention binds to all of residues 369-379, 389, 391-401, 403, and 418 of the D chain of human complement C3c set forth in SEQ ID NO:48.

[0084] The sequences of SEQ ID NOs: 47 and 48 are shown in Table 5 below. [Table 5]

[0085] It is highly likely that specific C3 epitope binding results in unexpected functional properties of the antibodies or antigen-binding fragments thereof of the present invention, such as high binding affinity to both C3 and C3b, and potent inhibition of all three pathways of complement activation, including the classical pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). therapeutic use In one aspect, the invention relates to an anti-C3 antibody, or an antigen-binding fragment thereof, for use as a medicament.

[0086] As already mentioned, we therefore show here that neutralization of C3 in the retina blocks the amplification loop of all complement pathways, thereby reducing the generation of cytotoxic membrane attack complex and proinflammatory complement components (C3a, C3b, iC3b, C5a) and improving clinical outcome in patients suffering from geographic atrophy. As a result, the present invention provides an anti-C3 antibody, or an antigen-binding fragment thereof, for use in the treatment or prevention of a retinal or ophthalmic disease.

[0087] In one aspect, the present invention provides an anti-C3 antibody or an antigen-binding fragment thereof for use in the treatment or prevention of an ophthalmic or ocular disease. In one aspect, the present invention provides an anti-C3 antibody or an antigen-binding fragment thereof for use in the treatment or prevention of an ophthalmic or ocular disease, including retinopathy, proliferative retinopathy (PR) such as retinopathy of prematurity, ischemic retinopathy, diabetic retinopathy (DR) including proliferative diabetic retinopathy (PDR) and non-proliferative diabetic retinopathy, diabetic macular edema (DME), diabetic macular ischemia (DMI), age-related macular degeneration (AMD) including dry AMD and wet AMD, geographic atrophy (GA), retinitis pigmentosa, inherited retinal dystrophies, myopic degeneration, retinal vein occlusion, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, any retinal disease. Provided is an anti-C3 antibody or an antigen-binding fragment thereof for use in the treatment or prevention of a disease selected from the group consisting of secondary choroidal neovascular membrane, optic neuropathy, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal neovascularization, polypoidal choroidal vasculopathy, retinal vasculitis, retinal microaneurysm, retrolental fibroplasia, chorioretinitis, Fuchs' dystrophy, macular telangiectasia, Usher syndrome, paroxysmal nocturnal hemoglobinuria (PNH), and Stargardt's disease.

[0088] In another embodiment, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof for use in the treatment or prevention of a disease selected from the group consisting of age-related macular degeneration, geographic atrophy, neovascular glaucoma, and diabetic retinopathy. In a further preferred embodiment, the present invention provides an anti-C3 antibody or antigen-binding fragment thereof for use in the treatment or prevention of geographic atrophy. In a preferred embodiment, the present invention provides an anti-C3 scFv for use in the treatment of geographic atrophy, comprising a variable heavy chain (VH) and a variable light chain (VL), wherein the VH comprises a CDR-H1 sequence of SEQ ID NO: 1, a CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2 and 15, a CDR-H3 sequence of SEQ ID NO: 3; and the VL comprises a CDR-L1 sequence of SEQ ID NO: 4, a CDR-L2 sequence selected from the group consisting of SEQ ID NO: 5 and 18, and a CDR-L3 sequence of SEQ ID NO: 6. In a particular embodiment, the anti-C3 scFv comprises a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO: 20 and SEQ ID NO: 21, respectively. In another particular embodiment, the anti-C3 scFv comprises a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO: 22 and SEQ ID NO: 23, respectively. In a further particular embodiment, the anti-C3 scFv comprises a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO: 24 and SEQ ID NO: 25, respectively.

[0089] The inventors have demonstrated that the anti-C3 antibody or antigen-binding fragment thereof according to the present invention exhibits even more advantageous properties than other "comparison compounds" targeting C3 described in the prior art and described in Example 5. The anti-C3 antibody or antigen-binding fragment thereof according to the present invention is distinct from therapeutic approaches based on APL2, NGM621 and ZIMURA®. APL-2 or pegcetacoplan is a pegylated derivative of the cyclic tridecapeptide compstatin, an inhibitor of complement component C3. The active component of APL-2 is APL-1. The inventors have developed alternative compounds (Comparative Compound A1 and Comparative Compound A2) for comparative purposes, which are illustrated in Example 5A. APL-2 has a large molecular weight equivalent to 350 kDa and a hydrodynamic radius of about 7.8 nm, which makes it difficult to penetrate deep into the retina. APL-2 has an effective duration of only one month, probably due to its low concentration of 3.5 mM. APL-2 is a PEGylated molecule, which increases its viscosity and makes it difficult to inject into the eye. Therefore, there is a need to more efficiently reduce the progression of GA.

[0090] NGM621 is a humanized IgG1 monoclonal antibody that strongly binds to complement C3 (C3) and inhibits complement activation. The inventors developed this compound for comparative purposes, as illustrated in Example 5B. NGM621 has been described as being able to inhibit both the classical and alternative pathways. ZIMURA® (or avacincaptad pegol) is designed to inhibit complement factor C5 cleavage into C5a and C5b. ZIMURA® does not bind C3. The inventors have illustrated in Example 6B that the format of the anti-C3 antibody or its antigen-binding fragment according to the present invention improves the amount of drug molecules delivered to the eye per intravitreal injection. In fact, it is illustrated that the scFv format can deliver a higher amount of drug per IVT injection compared to other compounds. This confirms that the scFv format can achieve greater complement inhibition and support therapeutic efficacy. In addition, the inventors have shown that the scFv format allows better retinal penetration after IVT injection compared to Fab or IgG.

[0091] As illustrated in Example 7, the inventors compared the potency of the anti-C3 antibodies or antigen-binding fragments thereof according to the present invention, particularly in comparison with compstatin derivatives such as comparative compound A1, and IgG directed against C3 such as comparative compound A3. The inventors showed that the antibodies of the present invention inhibit the classical, alternative, and lectin pathways more potently than comparative compound A1 and comparative compound A3. The inventors further evaluated that the exemplary antibodies of the present invention or fragments thereof are more potent in inhibiting CP and LP when compared to comparative compound A1. The inventors also showed that AP is improved in hemolysis assay in potency of inhibition compared to comparative compound A1. The present inventors also illustrated in Example 8 the binding affinity of the anti-C3 antibody or its antigen-binding fragment according to the present invention, compared to existing compounds, compstatin derivatives such as comparative compound A1, and IgG directed against C3 such as comparative compound A3. The high binding affinity of the exemplary antibody contributes to the prolongation of the neutralization time of C3 after intravitreal injection, further allowing for a reduction in injection frequency. The improved binding affinity and reduced injection frequency significantly improve the efficacy of the treatment of patients. It also provides valuable benefits to patients, in particular improved adherence and compliance with the drug.

[0092] In addition, the inventors have shown that the anti-C3 antibodies or antigen-binding fragments thereof according to the invention bind to human C3 and C3b with higher affinity, allowing for more potent inhibition of C3, compared to compstatin derivatives such as comparative compound A1 and IgG directed against C3 such as comparative compound A3. Compared to comparative compound A3, the antibodies of the invention bind to C3b with higher affinity, thereby blocking the generation of C3b in the C3 amplification loop and the activity of C3b and C3b already deposited and generated by the alternative activation pathway. The present inventors also demonstrated in Example 8 that the anti-C3 antibody or antigen-binding fragment thereof according to the present invention binds to a disease-associated single nucleotide polymorphism (SNP) of human C3, ensuring that the antibody of the present invention can treat a wide range of patients suffering from ophthalmic or ocular diseases. The present inventors further demonstrated that the binding affinity is improved compared to the binding affinity of comparative compound A1 to the SNP of human C3.

[0093] The present inventors demonstrate in Example 10 that the anti-C3 antibody or antigen-binding fragment thereof according to the present invention can improve permeation across Bruch's membrane, while comparative compounds A2 and ZIMURA® showed no penetration, and comparative compound A3 showed only reduced penetration. Since most complement activity is in the RPE / Bruch's membrane / CC region, good tissue penetration and diffusion through Bruch's membrane is essential for therapeutic approaches that inhibit the C3 pathway. Finally, the anti-C3 antibodies or antigen-binding fragments thereof according to the invention inhibit all complement effectors with high affinity and potency as exemplified in Example 7. This results in inhibition of the C3 amplification loop in all pathways as well as the activity of already formed C3b and the activity of C3b generated by C3 "tick-over" activation. The result is a complete inhibition of all complement activation pathways and all complement effector functions.

[0094] In conclusion, the present inventors have developed a highly promising therapeutic strategy for treating patients suffering from GA and have demonstrated the superiority of the anti-C3 antibody or its antigen-binding fragment according to the present invention over comparative compounds A2 and A3. The present inventors have demonstrated that the anti-C3 antibody or its antigen-binding fragment according to the present invention: Allows for improved delivery of drug per intravitreal injection (Example 6B), notably exhibiting good retinal penetration after equimolar intravitreal injection due to its format; · has an improved duration of therapeutic efficacy due, inter alia, to its format, ensuring improved dosing frequency and enhanced patient compliance; · Has good penetration into target tissues (Example 10), with the ability to penetrate the RPE and Bruch's membrane, thereby improving its therapeutic potential in the treatment of ophthalmic or eye diseases; have improved potency and more effective complement blockade, confirming that the anti-C3 antibodies or antigen-binding fragments thereof according to the invention inhibit all complement effector functions with high affinity and potency (Examples 7 and 8); · Has improved binding affinity for C3, contributing to an increased duration of C3 neutralization following intravitreal injection, further allowing for reduced injection frequency (Example 8); have an improved binding affinity for C3b, in particular, the anti-C3 antibody or antigen-binding fragment thereof according to the invention binds with similar affinity to C3 and C3b, thereby ensuring the inhibition of the generation of C3b in the C3 amplification loop (Example 8); and have improved binding affinity to disease-associated SNPs of human C3, ensuring that the antibodies of the invention can treat a variety of patients suffering from ophthalmic or eye diseases (Example 8); exhibiting improved stability (Examples 11 and 13); and Has a low immunogenicity risk (Examples 4 and 12) This was actually demonstrated.

[0095] In addition, in a particular embodiment of the present invention, the anti-C3 antibody or antigen-binding fragment thereof is not PEGylated. In this embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention exhibits a reduced risk of inducing angiogenesis, as observed in the disclosed clinical trials for comparative compounds such as ZIMURA® or APL-2. Overall, the antibodies of the invention have proven to be a highly effective treatment in wet AMD, particularly GA, with the longest duration of effect, the best improvement in patient compliance per mole of drug, and the best retinal efficacy. In one aspect, the invention provides a pharmaceutical composition comprising an anti-C3 antibody or antigen-binding fragment thereof and a pharma- ceutically acceptable carrier. The anti-C3 antibody or antigen-binding fragment thereof is administered by any suitable means, including intravitreal, oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the anti-C3 antibody is suitably administered by pulse infusion, particularly with decreasing doses of the antibody. In one aspect, dosing is given by injection, most preferably intravenous or subcutaneous injection, depending in part on whether the administration is brief or chronic. In one embodiment, the anti-C3 antibody is administered to the eye via intravitreal injection.

[0096] For the prevention or treatment of disease, the appropriate dosage of antibody, as defined above, will depend on a variety of factors, such as the type of disease being treated, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the patient's medical history and response to the antibody, and the judgment of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. In some embodiments, the applicable dose range of the antibody of the invention per injection is 1 mg / eye to 20 mg / eye, or between 5 mg / eye to 20 mg / eye, or between 10 mg / eye to 15 mg / eye, or about 15 mg / eye. The term "suppress" is used herein in the same context as "amelioration" and "alleviation" to mean the alleviation or reduction of one or more characteristics of a disease. The antibody compositions are formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration and other factors known to physicians. The "therapeutically effective amount" of the antibody to be administered will be determined by such considerations and is the minimum amount necessary to prevent, ameliorate or treat the ophthalmic disorder or eye disease addressed by the antibody of the invention.

[0097] The antibody need not be, but may be, formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of anti-C3 antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and routes of administration as used herein above, or at about 1-99% of the dosages previously used. In a specific embodiment, the present invention also provides a pharmaceutical composition comprising an anti-C3 antibody or antigen-binding fragment thereof and a pharma- ceutically acceptable carrier and at least one additional therapeutic agent. Treatment Method In another aspect, the present invention also encompasses any method for treating or preventing an ophthalmic or eye disease in a patient in need thereof, said method comprising administration of an anti-C3 antibody or antigen-binding fragment thereof of the present invention.

[0098] In some embodiments, the invention relates to a method for treating or preventing an ophthalmic or eye disease comprising administering a pharma- tically effective amount of an antibody or fragment thereof according to the invention to a patient in need thereof. In one embodiment, the disease is selected from the group consisting of retinopathy, proliferative retinopathy (PR), such as retinopathy of prematurity, ischemic retinopathy, diabetic retinopathy (DR), including proliferative diabetic retinopathy (PDR) and non-proliferative diabetic retinopathy, diabetic macular edema (DME), diabetic macular ischemia (DMI), age-related macular degeneration (AMD), including dry AMD and wet AMD, geographic atrophy (GA), retinitis pigmentosa, inherited retinal dystrophies, myopic degeneration, retinal vein occlusion, retinal artery occlusion, endophthalmitis, bud The present invention relates to a retinal vasculopathy, a retinal vascular endothelial disorder ... All disclosed technical features described herein are applicable to said methods of treatment.

[0099] Pharmaceutical Compositions and Their Administration The composition comprising the anti-C3 antibody or antigen-binding fragment thereof may be administered to a subject having or at risk of having an ophthalmic or ocular disease. The present invention further provides for the use of the anti-C3 antibody or antigen-binding fragment thereof in the manufacture of a medicament for the prevention or treatment of a C3-related disease. As used herein, the term "subject" refers to any mammalian patient, including, for example, humans and non-human mammals such as primates, rodents and dogs, to which the anti-C3 antibody or antigen-binding fragment thereof may be administered. Subjects specifically intended for treatment using the methods described herein include humans. The anti-C3 antibody or antigen-binding fragment thereof may be administered either alone or in combination with other compositions. Various delivery systems are known and can be used to administer anti-C3 antibodies or antigen-binding fragments thereof. Methods of introduction include, but are not limited to, intravitreal, eye drops, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural and oral routes. Anti-C3 antibodies or antigen-binding fragments thereof can be administered, for example, by infusion, bolus or injection, and can be administered together with other biologically active agents. Administration can be systemic or local. In a preferred embodiment, administration is by intravitreal injection. Formulations for such injections can be prepared, for example, in pre-filled syringes.

[0100] In certain embodiments, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention is administered intraocularly. Delivery of therapeutic compounds to various structures of the eye, such as the retina, is a challenge. Challenges include, but are not limited to, several restrictive ocular barriers, tear mechanisms including blinking and washing away of delivered compounds, limited local injection volume, limited local bioavailability, and low tolerance to impurities and contaminants (see, e.g., Patel et al. World J Pharmacol. 2013; 2(2): 47-64; Morrison et al. Ther. Deliv. 2014; 5(12): 1297-1315). The antibodies of the present invention may overcome these challenges. The antibodies of the present invention preferably have a molecular weight of about 60 kDa or less, preferably about 25 kDa. Examples of antigen-binding proteins of about 60 kDa or less include, but are not limited to, scFv, VHH, and Fab fragments. Examples of antigen-binding proteins of about 25 kDa or less include, but are not limited to, scFv and VHH. The smaller size of scFv allows for the delivery of more therapeutic compound per injection. This allows for a high concentration of antibody to the eye. The smaller size of the antibodies of the present invention, especially scFv, can also improve their penetration into disease-related tissues, i.e., the choroidal region of the eye. The antibodies of the present invention can penetrate one or more layers of the choroidal region, including Hurler's layer, Sattler's layer, choriocapillaris, and Bruch's membrane, thereby targeting complement C3 and C3b in these layers of the choroidal region.

[0101] In certain embodiments, intraocular administration is accomplished using a drug delivery device, such as a suprachoroidal drug delivery device or a subretinal drug delivery device. Suprachoroidal administration procedures involve administration of a drug to the suprachoroidal space of the eye and are typically performed using a suprachoroidal drug delivery device, such as a microinjector equipped with a microneedle (see, e.g., Hariprasad, Retinal Physician; 2016; 13: 20-23; Goldstein, 2014, Retina Today 9(5): 82-87, each of which is incorporated by reference in its entirety). In certain embodiments, intraocular administration is accomplished via the intravitreal route, which is often performed using a syringe and a 27-30 gauge needle (see, e.g., Jiang et al., supra). While all these forms of administration are clearly contemplated as being within the scope of the present invention, the form of administration is a solution for injection, particularly for intravitreal injection. Typically, suitable pharmaceutical compositions for injection may include a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), and optionally a stabilizer (e.g., human albumin). However, in other methods consistent with the teachings of the present specification, the antibody of the present invention may be delivered directly to the site of harmful cell populations, thereby increasing the exposure of diseased tissue to the therapeutic agent.

[0102] In an alternative embodiment, the pharmaceutical composition may be formulated according to routine procedures as a pharmaceutical composition adapted for intravenous or subcutaneous administration to humans. Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. Optionally, the medicament may also include a solubilizing agent and a local anesthetic, such as lignocaine, to ease pain at the site of the injection. Generally, the ingredients are supplied separately or mixed together in unit dosage form, for example as a dry lyophilized powder or water-free concentrate, in a hermetically sealed container, such as an ampoule or sachette indicating the quantity of active agent. When the medicament is administered by injection, it may be dispensed into an injection bottle containing sterile pharmaceutical grade water or saline. When the medicament is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration. Further, the pharmaceutical composition may be provided as a pharmaceutical kit comprising (a) a container containing the anti-C3 antibody or antigen-binding fragment thereof according to the present invention in lyophilized form, and (b) a second container containing a pharma- ceutically acceptable diluent for injection (e.g., sterile water). The pharma-ceutically acceptable diluent may be used for reconstitution or dilution of the lyophilized anti-C3 antibody or antigen-binding fragment thereof. Optionally associated with such container may be labeling in a form prescribed by a governmental agency regulating the manufacture, use, and sale of pharmaceuticals or biological products, which labeling reflects approval by the agency of manufacture, use, or sale for human administration.

[0103] The amount of the anti-C3 antibody or its antigen-binding fragment according to the present invention that is effective in treating or preventing ophthalmic or ocular diseases can be determined by standard clinical techniques. In addition, in vitro assays can be used to help identify optimal dosage ranges. The exact dose that can be used in the formulation also depends on the route of administration and the stage of the disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective amounts can be estimated from dose-response curves derived from in vitro or animal model test systems. For example, the toxicity and therapeutic efficacy of the anti-C3 antibody or antigen-binding fragment thereof according to the present invention may be improved by the ED 50(the dose that is therapeutically effective in 50% of the population) can be determined in cell cultures or experimental animals by standard pharmaceutical procedures. Anti-C3 antibodies or antigen-binding fragments thereof that exhibit larger therapeutic indices are preferred. The data obtained from cell culture assays and animal studies can be used to define a range of dosage for use in humans. The dosage of the anti-C3 antibody or antigen-binding fragment thereof is adjusted to an ED that produces little or no toxicity. 50 The circulating concentration of the antibody or antigen-binding fragment thereof is typically within a range including the IC50 / IC10 determined in cell culture. Dosages may vary within this range depending on the dosage form used and the route of administration utilized. For any anti-C3 antibody or antigen-binding fragment thereof used in the method, the therapeutically effective dose can be estimated initially from cell culture assays. Dosages are determined based on the IC50 / IC10 determined in cell culture. 50 The compound can be formulated in animal models to achieve a circulating plasma concentration range that includes the concentration of the test compound that achieves a half-maximal inhibition of symptoms (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography, ELISA, etc.

[0104] For intravitreal injection of the anti-C3 antibody or antigen-binding fragment thereof according to the present invention, longer intervals between treatments are generally preferred. Due to its improved potency, the anti-C3 antibody of the present invention can be administered at longer intervals. In one embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention is administered every 6 weeks, preferably every 7 weeks, preferably every 8 weeks, preferably every 9 weeks, preferably every 10 weeks, preferably every 11 weeks, more preferably every 12 weeks. In a further preferred embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention is administered once every 3 months. Since the volume that can be administered to the eye is strictly limited, it is very important that the anti-C3 antibody or antigen-binding fragment thereof according to the present invention can be formulated to a high concentration.Furthermore, the potency of the anti-C3 antibody or antigen-binding fragment thereof according to the present invention is very important, since a strong antibody can exert its effect even at a low dose, thereby extending the activity and the interval between treatments.

[0105] The antibodies of the present invention may be formulated in very high doses, including but not limited to 20mg / ml, 30mg / ml, 40mg / ml, 50mg / ml, 60mg / ml, 70mg / ml, 80mg / ml, 90mg / ml, 100mg / ml, 110mg / ml, 120mg / ml, 130mg / ml, 140mg / ml, 150mg / ml, 160mg / ml, 170mg / ml, 180mg / ml, 190mg / ml, 200mg / ml. Preferably, the anti-C3 antibodies or antigen-binding fragments thereof according to the present invention may be formulated in a liquid formulation at a concentration comprised between about 90mg / ml and 180mg / ml, more preferably between 130mg / ml and 160mg / ml. Preferably, the anti-C3 antibodies or antigen-binding fragments thereof according to the present invention may be formulated in a liquid formulation at about 150mg / ml. A typical dosage that can be administered to a patient is between about 2.5 mg / eye and 20 mg / eye, or between 7.5 mg / eye and 15 mg / eye. In a specific embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention can be administered to a patient at a concentration of 7.5 mg per eye. In another specific embodiment, the anti-C3 antibody or antigen-binding fragment thereof according to the present invention can be administered to a patient at a concentration of 15 mg per eye. Typical buffer components that can be used for such formulations include, for example, sodium acetate, PS20, and trehalose dihydrate.

[0106] In some embodiments, a pharmaceutical composition comprising an anti-C3 antibody or antigen-binding fragment thereof may further comprise a therapeutic agent, either conjugated or unconjugated to a binding agent. With respect to treatment regimens for combinatorial administration, in a specific embodiment, the anti-C3 antibody or antigen-binding fragment thereof is administered simultaneously with the therapeutic agent. In another specific embodiment, the therapeutic agent precedes or follows administration of the anti-C3 antibody or antigen-binding fragment thereof by at least one hour up to several months, e.g., at least one hour, five hours, twelve hours, one day, one week, one month, or three months prior to or following administration of the anti-C3 antibody or antigen-binding fragment thereof. Polynucleotides, Vectors, Host Cells and Recombinant Methods In one aspect, the invention encompasses isolated polynucleotides comprising sequences encoding anti-C3 antibodies or antigen-binding fragments thereof according to the invention, vectors and host cells comprising the polynucleotides, and recombinant techniques for producing antibodies. The isolated polynucleotides can encode any desired form of anti-C3 antibodies or antigen-binding fragments thereof, including, for example, full-length monoclonal antibodies, Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0107] A polynucleotide comprising a sequence encoding an anti-C3 antibody or an antigen-binding fragment thereof according to the present invention may be fused to one or more control or regulatory sequences known in the art and may be contained in a suitable expression vector or host cell known in the art. Each polynucleotide molecule encoding a heavy or light chain variable domain may be fused independently to a polynucleotide sequence encoding a constant domain, such as a human constant domain, to allow for the production of an intact antibody. Alternatively, polynucleotides or portions thereof may be fused together to provide a template for the production of a single chain antibody. For recombinant production, a polynucleotide encoding the antibody or antigen-binding fragment thereof is inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Many suitable vectors for expressing recombinant antibodies are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0108] The anti-C3 antibody or antigen-binding fragment thereof according to the present invention may also be produced as a fusion polypeptide in which the antibody or fragment thereof is fused to a heterologous polypeptide, such as a signal sequence or other polypeptide having a specific cleavage site at the amino terminus of the mature protein or polypeptide. The heterologous signal sequence selected is typically one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the anti-C3 antibody signal sequence, the signal sequence may be replaced by a prokaryotic signal sequence. The signal sequence may be, for example, the alkaline phosphatase, penicillinase, lipoprotein, heat-stable enterotoxin II leader, or the like. For yeast secretion, the native signal sequence may be replaced with a leader sequence obtained, for example, from yeast invertase alpha factor (including Saccharomyces and Kluyveromyces alpha-factor leaders), acid phosphatase, C. albicans glucoamylase, or the signals described in WO90 / 13646. In mammalian cells, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, may be used. DNA for such precursor region is ligated in reading frame to DNA encoding the humanized anti-C3 antibody, or antigen-binding fragment thereof.

[0109] Expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Generally, in cloning vectors, this sequence enables the vector to replicate independently of the host chromosomal DNA and includes an origin of replication or an autonomously replicating sequence. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2-v plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV and BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter). Expression and cloning vectors may contain a gene encoding a selectable marker to facilitate identification of expression. Typical selectable marker genes confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate or tetracycline, or alternatively encode a protein that complements an auxotrophic deficiency or supplies a particular nutrient that is otherwise absent in complex media, e.g., the gene encoding D-alanine racemase for Bacillus.

[0110] One example of a selection scheme utilizes drugs to stop the growth of host cells. Those cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin. Common selectable markers for mammalian cells are those that allow the identification of cells that are competent to take up nucleic acid encoding a humanized anti-C3 antibody, such as DHFR (dihydrofolate reductase), thymidine kinase, metallothionein-I and II (such as primate metallothionein genes), adenosine deaminase, ornithine decarboxylase, etc. Cells transformed with a DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. When wild-type DHFR is used, a suitable host cell is a Chinese hamster ovary (CHO) cell line that is deficient in DHFR activity (e.g., DG44). Alternatively, host cells transformed or co-transformed with DNA sequences encoding anti-C3 antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) (particularly wild-type hosts containing endogenous DHFR) can be selected by growing the cells in medium containing a selection agent for the selectable marker, such as an aminoglycoside antibacterial drug, e.g., kanamycin, neomycin, or G418. See, e.g., U.S. Patent No. 4,965,199.

[0111] When recombinant production is carried out in yeast cells as host cells, the TRP1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature 282: 39) can be used as a selectable marker. The TRP1 gene provides a selection marker for mutant strains of yeast lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics 85:12). The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2p-deficient yeast strains, such as ATCC 20,622 and 38,626, are complemented by known plasmids carrying the LEU2 gene. In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used for transformation of Kluyveromyces yeast. Alternatively, an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis (Van den Berg, 1990, Bio / Technology 8:135). A stable multicopy expression vector for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces has also been disclosed (Fleer et al., 1991, Bio / Technology 9:968-975).

[0112] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid molecule encoding the anti-C3 antibody or its polypeptide chain. Suitable promoters for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Promoters for use in bacterial systems also contain a Shine-Dalgamo (SD) sequence operably linked to the DNA encoding the humanized anti-C3 antibody. Many eukaryotic promoter sequences are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the site where transcription is initiated. Another sequence found 70-80 bases upstream from the start of transcription of many genes is a CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence, which may be a signal for addition of a poly A tail to the 3' end of the coding sequence. All of these sequences are suitable for insertion into eukaryotic expression vectors.

[0113] Examples of suitable promoting sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Inducible promoters have the added advantage of transcription being regulated by growth conditions. These include yeast promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, derivatives of enzymes involved in nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes involved in maltose and galactose utilization. Yeast enhancers are also advantageously used with yeast promoters. Transcription of the anti-C3 antibody or antigen-binding fragment thereof according to the present invention from a vector in a mammalian host cell is regulated by a promoter derived from the genome of a virus, such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40), from a heterologous mammalian promoter, for example, an actin promoter or an immunoglobulin promoter, or from a heat shock promoter, provided that such a promoter is compatible with the host cell system.

[0114] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., 1982, Nature 297:598-601, which discloses the expression of human p-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous sarcoma virus long terminal repeat can also be used as a promoter.

[0115] Another useful element that can be used in a recombinant expression vector is an enhancer sequence, which is used to increase the transcription of DNA encoding the anti-C3 antibody or antigen-binding fragment thereof according to the present invention by higher eukaryotes. A large number of enhancer sequences are now known from mammalian genes (e.g., globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, enhancers from eukaryotic cell viruses are used. Examples include the SV40 enhancer behind the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer behind the replication origin and adenovirus enhancers. See also Yaniv, 1982, Nature 297:17-18 for a description of enhancing elements for the activation of eukaryotic promoters. The enhancer can be spliced ​​into the vector at a 5' or 3' position of the anti-C3 antibody coding sequence, but is preferably located at a site 5' from the promoter. Expression vectors used in eukaryotic host cells (nucleated cells from yeast, fungi, insects, plants, animals, humans or other multicellular organisms) may also contain sequences necessary for the termination of transcription and for stabilizing mRNA. Such sequences are commonly available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-C3 antibody or antigen-binding fragment thereof according to the invention. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vectors disclosed therein. In some embodiments, anti-C3 antibodies can be expressed using the CHEF system (see, for example, U.S. Pat. No. 5,888,809).

[0116] Suitable host cells for cloning or expressing DNA in the vectors herein are prokaryotes, yeast or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include eubacteria such as gram-negative or gram-positive bacteria, such as Escherichia coli, such as E. coli, Enterobacter, Erwinia, Klebsilea, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans and Shigella, and bacilli, such as B. subtilis and B. licheniformis (e.g. B. licheniformis 41P, disclosed in DD 266,710 published April 12, 1989), Pseudomonas, such as P. aeruginosa and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative and not limiting.

[0117] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-C3 antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, Schizosaccharomyces pombe; Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia partoris; pastors (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and numerous other genera, species and strains of filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. niger, are commonly available and useful herein.

[0118] Suitable host cells for the expression of glycosylated anti-C3 antibodies or antigen-binding fragments thereof are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells, including many baculovirus strains and variants and corresponding permissive insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori (silkworm). Various virus strains for transfection are publicly available; for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, such viruses can be used specifically for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be utilized as hosts. The anti-C3 antibody or its antigen-binding fragment according to the present invention may be incorporated into a viral vector, i.e., a polynucleotide encoding the anti-C3 antibody or its antigen-binding fragment is introduced into a viral vector and then expressed in the patient's body after infection with the virus.

[0119] In another embodiment, expression of the anti-C3 antibody or antigen-binding fragment thereof according to the invention is carried out in vertebrate cells. Propagation of vertebrate cells in culture (tissue culture) has become a routine procedure and techniques are widely available. Examples of useful mammalian host cell lines are SV40 transformed monkey kidney CV1 line (COS-7, ATCC CRL1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, (Graham et al., 1977, J. Gen Virol. 36: 59), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR1 (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4216; e.g., DG44), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat hepatocytes (BRL 3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human hepatocytes (Hep G2, HB8065), mouse mammary tumor (MMT060562, ATCC CCL51), TR1 cells (Mather et al., 1982, Annals NY Acad. Sci. 383: 44-68), MRC5 cells, FS4 cells, and a human hepatoma line (HepG2).

[0120] Host cells are transformed with the expression or cloning vectors described above for producing anti-C3 antibodies or antigen-binding fragments thereof and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants or amplifying genes encoding the desired sequences.

[0121] The host cells used to produce the anti-C3 antibody or antigen-binding fragment thereof according to the present invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma-Aldrich Co., St. Louis, Mo.), Minimal Escherichia coli (MEM), (Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.) and Dulbecco's Modified Eagle's Medium (DMEM), Sigma-Aldrich Co. are suitable for culturing the host cells. In addition, the following media are described in Ham et al., 1979, Meth. Enz. 58: 44; Barnes et al., 1980, Anal. Biochem. 102: Any medium described in one or more of US Patent Nos. 255, 4,767,704, 4,657,866, 4,927,762, 4,560,655, 5,122,469, WO90 / 103430 and WO87 / 00195 may be used as a culture medium for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) and glucose or an equivalent energy source. Other supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those previously used with the host cell selected for expression and will be apparent to those skilled in the art.

[0122] When using recombinant techniques, the antibody or fragment thereof may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step the cells may be disrupted to release the protein. Particulate debris, host cells or lysed fragments may be removed, for example, by centrifugation or ultrafiltration. Carter et al., 1992, Bio / Technology 10:163-167, describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris may be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration device. Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.Various methods may be used to isolate antibodies from host cells.

[0123] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being a typical purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (see, for example, Lindmark et al., 1983 J. Immunol. Meth. 62:1-13). Protein G is recommended for all mouse isotypes and for human gamma 3 (see, for example, Guss et al., 1986 EMBO J. 5:1567-1575). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. H3 For antibodies containing the IgG1 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0124] Following any preliminary purification steps, the mixture containing the antibody of interest or fragment thereof and contaminants may be subjected to low pH hydrophobic interaction chromatography, typically performed at low salt concentrations (e.g., about 0-0.25 M salt) using an elution buffer at a pH between about 2.5-4.5. Also included are nucleic acids that hybridize under low, medium and high stringency conditions as defined herein to all or part of the nucleotide sequence represented by the isolated polynucleotide sequence encoding an anti-C3 antibody or antigen-binding fragment thereof according to the present invention (e.g., the portion encoding the variable region). The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (e.g., 20, 25, 30 or 50) nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least 80%, e.g., at least 90%, at least 95% or at least 98% identical to a portion or all of the sequence of the nucleic acid encoding an anti-C3 polypeptide (e.g., heavy or light chain variable region) or its complement. Hybridizing nucleic acids of the type described herein can be used, for example, as cloning probes, primers, e.g., PCR primers or diagnostic probes.

[0125] In one embodiment, the invention relates to an isolated polynucleotide or polynucleotides comprising a sequence encoding a heavy chain variable region as set forth in SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24, and a sequence encoding a light chain variable region as set forth in SEQ ID NO:21, SEQ ID NO:23 and / or SEQ ID NO:25. In the anti-C3 antibodies and antibody fragments, the nucleic acid sequences encoding the CDRs remain unchanged (unchanged with respect to the amino acids they encode, equivalent DNA sequences are possible due to codon degeneracy), but it is understood that the surrounding regions, e.g., the FR regions, may be manipulated. manufactured goods In another embodiment, an article of manufacture is included that contains materials useful for the treatment of the disorders described above. The article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition that is effective for treating a condition and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle. The active agent in the composition is an anti-C3 antibody or an antigen-binding fragment thereof according to the present invention. A label on or associated with the container indicates that the composition is used for treating a selected condition. The article of manufacture may further include a second container that contains a pharma- ceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use. The present invention is further described in the following examples, which are not intended to limit the scope of the invention. EXAMPLES

[0126] Example 1 C3 mechanisms and the pathogenesis of geographic atrophy In patients with GA, increased and uncontrolled activation of the complement system leads to destruction of the choriocapillaris, damage to RPE cells and ultimately loss of photoreceptors. The relevance of complement activation with respect to the pathophysiology of diseases is based on the following: A. Expression of complement proteins and activation of the complement system in the retina of AMD patients All proteins of the complement cascade were found to be expressed in various cell types locally in the retina. In patients, the protein levels of several complement factors (e.g., C3, CFB, and C5) were increased in drusen and GA lesions. In addition, increased levels of complement activation products C3a, C4a, C3d, and CBb were found to be increased in the aqueous humor and plasma of AMD patients. Immunohistochemical staining for membrane attack complex (MAC) shows increased deposition in Bruch's membrane, RPE layer, and choriocapillaris near GA lesions in patients.

[0127] B. Genetics of AMD GWAS studies have identified the Y402H polymorphism in complement factor H (CFH) as a major risk factor for developing AMD. An increased risk of up to 7-fold was found in individuals homozygous for this mutation. CFH is the main negative regulator of the complement system. It enhances the decay of the C3 convertase C3bBb and controls the proteolytic degradation of already deposited C3b. The Y402 polymorphism results in reduced binding of CFH to other proteins and increased levels of complement activation products, as well as increased deposition of MAC was found in homozygous patients. In addition, polymorphisms in other complement genes (e.g., CFB, C3, C7, C9) have been associated with the development of AMD. C. Clinical Studies with Complement Inhibitors Existing therapeutic strategies, including treatment with C3 inhibitors (APL-2) and C5 inhibitors (ZIMURA®), have reduced the growth of GA lesions in Phase 2 clinical trials. Inhibition of C3 inhibits the C3 amplification loop, thereby inhibiting the alternative, classical and lectin pathways of the complement system.

[0128] Example 2 Generation and characterization of anti-C3 antibody library To generate anti-C3 antibodies, three New Zealand White rabbits are immunized with native human C3 protein. Each animal receives four injections of C3 protein at various times with Freund's complete or incomplete adjuvant. The immune response of each animal is tested using ELISA, and specific antibody titers in the serum indicate a good immune response. An scFv antibody cDNA library is constructed via PCR amplification from RNA extracted from isolated rabbit PBMCs and splenic lymphocytes. The coding sequences for the variable light and heavy domains are amplified separately and linked through a series of overlapping PCR steps to yield the final scFv product. The amplified DNA sequence encoding the rabbit-derived scFv is digested with appropriate restriction enzymes and ligated into a phagemid vector, which is transformed into E. coli TG1 electrocompetent cells, which are well suited for antibody phage display library generation.

[0129] Example 3 Screening for anti-C3 antibodies that inhibit all three complement pathways To screen for anti-C3 antibodies with high affinity, the scFvs displayed on the phage were subjected to several rounds of biopanning (selection) against native human C3. The stringency of the selection is increased in each round by either decreasing the concentration of C3 protein used in biopanning or increasing the stringency of the washes. Approximately 380 monoclonal phages are selected and screened for their ability to bind to C3 in an ELISA assay. Based on the phage ELISA data and DNA fingerprinting, a selection of the displayed scFvs are recombinantly produced as antibody proteins in the Fab format. The resulting proteins are assessed for their ability to bind human C3 and C3b.

[0130] To identify antibodies that block all three complement pathways, the antibodies are screened using an enzyme immunoassay for the qualitative determination of functional classical, lectin and alternative complement pathways in human serum using the WEISLAB® Complement system Screen (Svar Life Science AB, Malmo, Sweden). As shown in Table 6, clone IV (SEQ ID NO: 32) was identified as being able to inhibit all three complement pathways. [Table 6]

[0131] The clone IV sequence is shown in Table 7 below. [Table 7]

[0132] Example 4 Reformatting, humanization and optimization operations Reformatting and humanization: For antibody humanization and reformatting to scFv antibody fragment format, rabbit-derived clone IV CDR sequences are grafted onto the light chain variable (VL) and heavy chain variable (VH) domains of human germline sequences. For VH humanization, the CDRs are grafted onto the IMGT_hVH_3_30 framework. For VL humanization, the CDRs are grafted onto the IMGT_hVL_4-69 framework. The variable domains are connected in a VL-linker-VH fashion using the (GGGGS)4 linker (SEQ ID NO: 46). For framework 4, IGLJ2 * 01 using the junction (J) gene. The reformatted and humanized variant clone V (SEQ ID NO: 33) retains its ability to inhibit the classical and alternative complement pathways, but exhibits lower binding affinity to C3 and C3b than the parent molecule clone IV. The variant was further modified by replacing additional rabbit-derived amino acids with the germline sequence, thereby generating clone VI (SEQ ID NO: 34). Its affinity for human C3 and C3b was comparable to the parent molecule, and the thermostability of the construct was increased by 11.7°C.

[0133] The clone V and clone VI sequences are summarized in Table 8 below. [Table 8]

[0134] affinity maturation Library construction: To improve the affinity of the molecule, a site-saturation library is constructed using clone VI (SEQ ID NO: 34) as a template. Randomization is performed on CDRs L1, L2, L3 and H3. Biopanning: Libraries containing light chain randomization are combined into one library. Similarly, libraries containing heavy chain randomization are combined into another library. Light and heavy chain libraries are subjected to biopanning against human C3. In rounds 2, 3 and 4 of biopanning (ROP2-4), a competitive effect at 50 μg / mL against clone IV is introduced to increase the stringency of selection, allowing the identification of binders with higher affinity than the parent molecule. Hits identified via phage ELISA in ROP2-4 are subjected to sequencing. The most promising candidates from ROP4 are expressed in scFv format and purified.

[0135] Hit characterization: All hits identified in ROP4 contain amino acid substitutions in CDR L3 or CDR H3. All hits were successfully expressed and purified. The best binding affinities for human C3 and C3b were observed for clone VII (SEQ ID NO:35), clone VIII (SEQ ID NO:36) and clone IX (SEQ ID NO:37), with C3 affinities of 1.1 nM, 184 pM and 1.3 nM, respectively.

[0136] Clone VII and Clone IX showed binding affinity and complement inhibitory activity comparable to the parent molecule, Clone IV. Clone VIII showed approximately a 10-fold increase in binding affinity to C3 and C3b compared to Clone IV, as well as superior performance in complement inhibition assays.

[0137] The sequences of clones VII, VIII and IX are summarized in Table 9 below. [Table 9]

[0138] Stabilization of scFv Stabilization of the scFv was achieved by the introduction of a disulfide bridge (VL 43C, VH 105C). Natural antibodies have a conserved interchain disulfide bridge between the constant light chain and the constant heavy chain CH1 domain, which strongly stabilizes the interaction between the light chain and the heavy chain. The absence of this interchain disulfide bond in the scFv may lead to variable domain destabilization and the formation of dimers, trimers and even higher oligomeric species, especially when formulated at high concentrations. Thus, the presence of a disulfide bond may stabilize the VH / VL interface of the scFv.

[0139] The stability of the heavy chain framework of clone VI was investigated by introducing framework substitutions, thereby generating inter alia clone X (SEQ ID NO: 38). Clone X and its disulfide bond stabilized variant clone XI (SEQ ID NO: 39) were compared in a stability study at 37° C. and protein concentrations >100 mg / mL. Incubation of clone XI at 37° C. for 2 weeks resulted in only a 3.3% decrease in monomer purity, while the parent molecule clone X experienced a 60.4% decrease in monomer content (Table 10). [Table 10]

[0140] The sequences of clones X and XI are summarized in Table 11 below. [Table 11]

[0141] Final optimization The most promising affinity maturation substitutions and disulfide bond stabilization were combined in clone XII (SEQ ID NO: 40), clone XIII (SEQ ID NO: 41) and clone XIV (SEQ ID NO: 42). The thermal stability and affinity for human C3 and C3b are determined by DSF and SPR assays. Clone XII, clone XIII and clone XIV showed melting temperatures of 70.4°C, 67.7°C and 67.2°C, respectively. The binding affinities for human C3 and C3b were 4.7nM and 4.8nM for clone XII, 116pM and 110pM for clone XIII, and 129pM and 116pM for clone XIV, respectively.

[0142] Clone XII, Clone XIII and Clone XIV are also tested for their activity in complement inhibition assay. The IC50 values ​​for complement inhibition in the classical pathway were 0.098 μM, 0.06 μM and 0.05 μM for Clone XII, Clone XIII and Clone XIV, respectively, while the parent Clone IV showed an IC50 of 0.08 μM. The IC50 values ​​for complement inhibition in the alternative pathway were 0.21 μM, 0.26 μM and 0.25 μM for Clone XII, Clone XIII and Clone XIV, respectively, while Clone IV showed an IC50 of 0.28 μM. In summary, Clone XIII and Clone XIV showed better functional properties than Clone IV. Additionally, Clone XIII and Clone XIV were compared in a concentration-dependent stability study. Clone XIII could be concentrated to 150 mg / mL without a decrease in monomer content, which decreased by only 2.2% after 2 weeks of incubation at 37°C, whereas clone XIV already lost 1.6% monomer content at concentration to 50 mg / mL, which decreased by a further 2.3% after 2 weeks of incubation at 37°C.

[0143] The sequences of clone XII, clone XIII and clone XIV are summarized in Table 12 below. [Table 12]

[0144] Final humanization: Immunogenicity risk prediction using EpiVax reveals a moderate risk for clone X (parent construct of clone XIII). EpiVax scores of -39.36 and 14.86 were determined for VH and VL, respectively. In addition, clone XI showed 77% human germline sequence identity for VH and 87% for VL. The evolution of clone IX to clone XIII involved exclusively CDR loop modifications, such that the trends identified in the FRs of clone XI remained unchanged in clone XIII. Therefore, additional germlining was performed in the framework regions. Additionally, computational analysis allows the identification of residues that are rarely present in the human antibody repertoire and therefore have increased immunogenicity risk, leading to the inclusion of the following mutations VH S61D, W62S, A63V and Y30S, and VL T56S, to further reduce the immunogenicity risk.

[0145] In the final humanization and optimization campaign, we generated clone III, which lacks important post-translational modification (PTM) motifs and was further humanized (86% and 89% human sequence identity for VL and VH, respectively) and affinity matured. In addition, an isomerization motif (DG) in CDR L2 was investigated since it had a large effect on refolding yield and affinity. Therefore, clone III was modified by introducing the mutation E50D, resulting in clone II, which showed 87% and 89% human sequence identity for VL and VH, respectively. An additional variant of clone II was generated by swapping the entire CDR H2 with the complete human germline sequence (increasing human sequence identity to human germline by an additional 7% in VH), thereby creating clone I. Thereby, the inventors have ensured that the anti-C3 of the present invention is not prone to potential isomerization, deamidation and oxidation.The inventors have further confirmed that the anti-C3 antibody or its antigen-binding fragment according to the present invention has acceptable viscosity, strong storage stability, high affinity for C3, C3b, shows similar potency in classical, alternative and lectin pathways, shows diffusion through porcine Bruch's membrane and does not show significant T cell activation.

[0146] Exemplary antibodies of the invention include: - Clone I: contains the variable heavy chain of SEQ ID NO: 20 and the variable light chain of SEQ ID NO: 21; - Clone II: containing the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 23; and - Clone III: comprises the variable heavy chain SEQ ID NO:24 and the variable light chain SEQ ID NO:25. immunogenicity The inventors further evaluated the immunogenicity of the scFvs of the invention.The inventors evaluated the predicted immunogenicity of exemplary antibodies according to the invention: Clone I, Clone II and Clone III.

[0147] To this end, we used a computational tool to predict such T cell epitopes (EpiMatrix, developed by EpiVax). By screening the sequences of a large number of human antibody isolates, EpiVax has identified several highly conserved HLA ligands that appear to have regulatory potential. Experimental evidence suggests that many of these peptides are actively tolerogenic in most subjects. These highly conserved, regulatory and promiscuous T cell epitopes are known as Tregitopes (De Groot et al. Blood. 2008 Oct 15;112(8):3303-11). The immunogenic potential of neoepitopes contained in humanized antibodies can be effectively modulated in the presence of a significant number of Tregitopes. For the purpose of antibody immunogenicity analysis, EpiVax includes a Tregitope-adjusted EpiMatrix score and a corresponding prediction of anti-therapeutic antibody response. To calculate the Tregitope-adjusted EpiMatrix score, the score of the Tregitope is subtracted from the EpiMatrix protein score. The Tregitope-adjusted score has been shown to correlate well with observed clinical immune responses for a set of 23 commercially available antibodies (De Groot et al. Clin Immunol. 2009 May;131(2):189-201).

[0148] The results on the EpiMatrix scale are summarized in Table 13 below. [Table 13] The sequences of the antibodies of the invention score lowest on the EpiMatrix scale, indicating that the scFvs of the invention have very limited potential for immunogenicity. Said EpiMatrix scale is well known to those skilled in the art and can be found, inter alia, in Figure 2 of the publication Mufarrege et al. Clin Immunol., 2017 Mar;176:31-41.

[0149] Example 5 Preparation of comparative compounds A. APL-1 surrogate and APL-2 surrogate For comparative purposes, the inventors have developed several derivatives of compstatin as follows: APL-1 surrogate: a PEGylated monocompstatin derivative (hereinafter designated as "comparative compound 1" or "compound A1") APL-2 surrogate: a PEGylated dimerized compstatin derivative (hereinafter designated as "Comparative Compound A2" or "Compound A2").

[0150] These compounds are disclosed as APL-1 and APL-2 in U.S. Patent Application Publication No. 20200282012. · Ricklin D, Lambris JD. Compstatin: a complement inhibitor on its way to clinical application. Adv Exp Med Biol. 2008;632:273-292. doi:10.1007 / 978-0-387-78952-1_20, and · Mastellos DC, Ricklin D, Lambris JD. Clinical promise of next-generation complementary therapeutics. Nat Rev Drug Discov. 2019;18(9):707-729. doi:10.1038 / s41573-019-0031-6 This is further disclosed in Compound A1 is an anti-C3 cyclic peptide derived from compstatin. Compound A2 is further developed based on Compound A1 to extend its circulating half-life and improve its solubility in serum. Compound A2 contains two peptide moieties linked via a linear 40kDa PEG linker.

[0151] The sequences of Compound A1 and Compound A2 are summarized in Table 14 below. [Table 14] Additional data regarding the structures and sequences of Compound A1 and Compound A2 is available in Adv Exp Med Biol. 2008; 632: 273-292.

[0152] B.NGM621 Surrogate For comparative purposes, the present inventors have developed an NGM compound disclosed in WO2019195136, preferably as an NGM621 surrogate, hereinafter referred to as "Comparative Compound A3" or "Compound A3". Compound A3 is an anti-C3 IgG with the following properties:

[0153] The related sequences are summarized in Table 15 below: [Table 15]

[0154] Based on the sequence described above, the inventors further developed a NGM621 surrogate in a Fab format, which compound is referred to as "Compound A3 Fab."

[0155] Example 6 Comparison Data A. Various mechanisms of action The exemplary antibodies of the present invention and the comparative compounds have different mechanisms of action and different properties, as summarized below: - the antibodies of the invention inhibit C3 and C3b, - APL-2 inhibits C3 and C3b; - NGM621 inhibits C3, and - ZIMURA® inhibits C5.

[0156] The inventors have shown that, in contrast to NGM621, an exemplary antibody of the invention, Clone I, binds to C3 and C3b with similar affinity, as illustrated in Table 16 below. [Table 16]

[0157] B. ScFv format can deliver more drug molecules per IVT injection The inventors have established the mass weights, injection volumes and possible doses of exemplary antibodies according to the invention and comparative compounds A2, A3 and ZIMURA® (data available in the literature), as illustrated in Table 17 below. [Table 17]

[0158] The antibodies of the invention have been shown to provide a higher drug dose per IVT administration to the eye, contributing to achieving greater complement inhibition in the eye. C. The ScFv format allows for better retinal penetration compared to Fab or IgG following equimolar intravitreal injection. We compared the retinal penetration of scFv (brolucizumab) and Fab (ranibizumab) after equimolar intravitreal injection in cynomolgus monkeys. The data show that the scFv format allows for improved retinal penetration compared to Fab or IgG. Thus, the antibodies of the present invention in scFv format are expected to achieve superior retinal exposure levels compared to Fab upon intravitreal administration (Figures 1A and 1B).

[0159] Example 7 Potency and in vitro activity of the scFv of the present invention and comparison with comparative compounds A1 and A3 To determine the potency of and comparison between the functions of exemplary scFvs of the present invention, the following assays are performed: a complement inhibition assay measuring the generation of the membrane attack complex and a hemolytic assay. A. Materials and Methods A. Complement inhibition assay To monitor the inhibition of the complement system in human serum, the scFv of the invention or comparative molecules are tested using an enzyme immunoassay using the WEISLAB® Complement system Screen (Svar Life Science AB, Malmo, Sweden) according to the supplier's instructions for the qualitative determination of functional classical, lectin and alternative complement pathways in human serum. The amount of C5b-C9 neoantigen produced is proportional to the functional activity of the complement pathway. The scFv of the invention and comparative molecules are tested in concentration response curves and the IC50 for inhibition is calculated using GraphPadPrism software and nonlinear 4-parameter curve regression.

[0160] B. Hemolysis assay Anti-C3 antibody fragments are evaluated for their ability to inhibit hemolysis of antibody-sensitized sheep red blood cells mediated by classical pathway (CP) or alternative pathway (AP) activation in human serum. For this, the surface of sheep red blood cells is coated with hemolysin (rabbit anti-sheep antibody) to activate the complement system in human serum, resulting in C3 activation and subsequent formation of the membrane attack complex (MAC). The MAC assembles with the membrane of sheep red blood cells, resulting in cell lysis. The level of hemoglobin released from lysed red blood cells is measured at OD414nm, which is proportional to the complement activity present in the serum sample. To determine the ability of anti-C3 molecules to inhibit CP activity, human serum is diluted in GVB++ buffer (Complement technology) for optimal activation of the classical pathway (requiring calcium and magnesium ions). Serial dilutions of anti-C3 test samples are prepared from this solution at a final concentration of 1% human serum for each data point (before addition to red blood cells). Samples are incubated with antibody-sensitized sheep red blood cells for 1 hour at 37° C. and released hemoglobin is measured in the supernatant after a centrifugation step. The alternative pathway (AP) is naturally activated by rabbit erythrocytes in human serum samples. Because AP activation requires high serum concentrations, the classical pathway needs to be blocked using MgEGTA to chelate Ca2+ ions (AP only requires Mg2+ ions). AP activation results in C3 activation and subsequent formation of the membrane attack complex (MAC). The MAC assembles on the membrane of rabbit erythrocytes, resulting in cell lysis. The level of hemoglobin released from lysed erythrocytes is measured at OD414nm and is proportional to the complement activity present in the serum sample.

[0161] B. Results The results are summarized in Table 18 below. [Table 18]

[0162] The exemplary antibodies of the invention (clone I, clone II and clone III) are more potent than compound A1 (the active component of compound A2). Importantly, the inventors have shown that the exemplary antibodies of the invention inhibit the classical and lectin pathways more potently than compound A1. Clone I exhibits activity in the alternative pathway similar to compound A1. Clone I exhibits more potent inhibition of the classical pathway compared to compound A1. Clone I shows more potent inhibition of the lectin pathway compared to compound A1. Clone I: The inventors have shown that an exemplary antibody of the invention (Clone I) inhibits all complement effector functions with high affinity and potency. Compound A1: The present inventors have demonstrated that comparative compound A1 inhibits all complement effector functions with lower affinity and potency when compared to the antibodies of the present invention. NGM621: We have shown that the NGM621 surrogate, comparator compound A3, does not exhibit C3b inhibition. ZIMURA®: The ZIMURA® compound targets only C5.

[0163] Example 8 Binding affinity to human C3 and C3b and comparison of the binding affinity of the antibody of the present invention, comparative compound A1, and comparative compound A3 The inventors evaluated the binding affinity of exemplary scFvs of the invention to human C3, and the binding affinity of Compound A1 and Compound A3 to human C3 and C3b. A. Materials and Methods The kinetic parameters of the candidates are determined using surface plasmon resonance (SPR) and a Biacore T100 device. C3 and its different variants (Table 19) are immobilized as ligands on a HC30M SPR sensor chip (XanTec bioanalytics, Germany) using EDC / sulfo-NHC linking chemistry. To determine the kinetic parameters, five increasing concentrations of each molecule (analyte) are injected sequentially using the single cycle kinetics (SCK) mode. Regeneration between candidates is performed using 10 mM glycine. * HCl, 2M NaCl, pH 3.5. Data were fit to a 1:1 Langmuir binding model to determine on-rates (ka), off-rates (kd) and affinities (K D The kinetic parameters of the reaction were determined.

[0164] Single nucleotide polymorphisms (SNPs) in C3 have been observed across populations, and certain SNPs have been identified in genome-wide association studies (GWAS) as associated with an increased risk of developing AMD. Common SNP variants include P314L, which is present in approximately 23.3% of the population, and R102G, which has been reported to be present in approximately 26-29% of the population. Very rare variants, present in less than 2.5% of the population, include K155Q, R735W, S1619W, K65Q, and R161W. Therefore, we measured the binding affinity of the scFv of the present invention to the most commonly occurring C3 forms, namely R102G and P314L. Wild-type human C3, human C3 P314L and human C3 R102G are expressed in a CHO cell line using the ExpiFectamine CHO Expression Kit (Thermo Fisher Scientific). Proteins are expressed to contain a 6xHis tag at the N-terminus and purified using a HisTrap Excel HP column (GE Healthcare) followed by a size exclusion chromatography step (SUPERDEX® 20010 / 300GL; GE Healthcare).

[0165] B. Results Kinetic and affinity data for the scFv of the invention, compound A1 (the active component of compound A2) and compound A3, which bind to human C3 and C3b, respectively, are listed in Table 19 below. [Table 19]

[0166] Compound A3 binds to C3 with high affinity to intact human C3 (KD=0.34 nM) but exhibits significantly lower affinity (>100-fold) to the C3 cleavage fragment C3b. Conversely, exemplary antibodies of the invention bind C3 with higher affinity than compound A3. In addition, exemplary antibodies of the invention have approximately equal binding affinity for C3 and C3b. It is noteworthy that the antibodies of the invention bind to human C3 and C3b with higher affinity compared to Compound A1 and Compound A3, allowing for more potent inhibition of C3. Compared to Compound A3, the antibodies of the invention bind to C3b with higher affinity, thereby blocking the generation of C3b in the C3 amplification loop as well as the activity of already deposited C3b and C3b generated by the alternative activation pathway.

[0167] Additional results are summarized in Table 20 below. [Table 20]

[0168] Thus, the inventors have confirmed that the antibodies of the present invention, including clone I, clone II and clone III, have higher affinity for C3 and C3b than compound A1 and more potently inhibit complement 2 / 3 activation. The high binding affinity of the exemplary antibody contributes to the extension of the time of neutralization of C3 after intravitreal injection, further allowing for a reduction in injection frequency. Improved binding affinity and reduced injection frequency significantly improve the efficacy of treatment for patients. This also provides valuable benefits to patients, particularly improved drug adherence and compliance. It is further noteworthy that the scFv of the present invention bind to wild-type C3 as well as the disease-associated SNP C3 P314L with higher affinity than compound A1.

[0169] Example 9 Identification of antibody-binding epitopes on human complement C3. Protein preparation: Complement component C3c is the major fragment resulting from C3 cleavage by C3 convertase and factor I. C3c isolated from human plasma is purchased from Athens Research & Technology (product number 16-16-030303), deglycosylated with PNGase F, and purified by size-exclusion chromatography on a SUPERDEX® 200 column. Peak fractions are pooled and concentrated using an Amicon filter device with a 50 kDa cutoff, and stored at -80°C. Crystallization of scFv: Crystals are obtained using the hanging drop vapor diffusion method by mixing 0.1 μl of protein with 0.1 μl of reservoir solution containing 0.1 M ammonium acetate, 0.1 M zinc chloride, 0.1 M BIS-TRIS pH 6.0 and 15% w / v PEG Smear High. Plate-like crystals appear within a day. Crystals are cryoprotected using reservoir solution supplemented with 30% v / v glycerol and flash frozen in liquid nitrogen.

[0170] Data collection and structure determination: Data are collected at 100K on a beamline X10SA at SLS, Villigen, Switzerland. 3600 frames are collected on an EIGER X16M detector at 0.1° per frame. Data are processed using autoPROC. Molecular replacement models are generated using SCULPTOR in the PHENIX software suite using a previously determined X-ray structure of a similar scFv as template. Molecular replacement is performed using PHASER using the scFv model generated using SCULPTOR as template. The structure is refined using Buster, model building and least squares analysis is performed using COOT. Cryo-EM sample preparation and data collection: C3c is mixed with ScFv in a 1:1.2 ratio, incubated at 277K for 16 hours, and purified using a SUPERDEX® 200 increase column. Peak fractions are concentrated to 36 μM and applied to EM-grids (Quantifoil-1.2 / 1.3 Au 300). Cryo-EM grids are made using a Leica GP (Leica Microsystems). Grids are blotted for 2 seconds at 4° C. and 85% humidity and placed in liquid ethane cooled by liquid nitrogen. Cryo-grids are screened on a Glacios microscope operated at a voltage of 200 kV with a Falcon III detector, and good quality grids are transferred to a Titan Krios microscope operated at 300 kV. Images are collected in super-resolution mode using a Falcon IV detector (Thermo Fisher Scientific) mounted behind an energy filter with a calibrated pixel size of 0.745 Å per pixel. Microscope image 60e - / Å 2 Expose with a total dose of 100 Å and process using CryoSPARC v3.1.1. Stacks are motion corrected and binned 2x to obtain a pixel size of 1.49 Å per pixel.

[0171] Cryo-EM image analysis: A total of 1,914,120 particles are picked from 3622 micrographs using the CryoSPARC template picker. Picked particles are extracted using a box size of 348 pixels and binned at 174 pixels in CryoSPARC. Extracted particles are then subjected to three rounds of 2D classification in cryoSPARC-3.1.1 to remove ice, contaminants and aggregates, resulting in 421,628 particles. Classes with well-defined C3c-scFv densities are used for ab initio reconstruction in cryoSPARC-3.1.0. 245,993 particles are selected based on a 2D class 0.9 threshold and subjected to one round of uniform and heterogeneous refinement. Particles are re-extracted using a box size of 348 pixels and two one further rounds of uniform and heterogeneous refinement are performed. This resulted in a fourier shell resolution of 2.55 Å. Model building: X-ray structures of C3c (pdb 5FOB) and scFv are integrated into EM densities using CHIMERA, real-space refined using PHENIX, and probed with COOT. Interface analysis is performed using PYMOL.

[0172] The structure of clone I is determined by X-ray crystallography at a resolution of 1.4 Å. The structure contains two molecules per asymmetric unit. All residues were well resolved in the electron density map, except for the two N-terminal residues of chains A and B, linker residues 115-132 of chain A and 114-133 of chain B, and the C-terminal residue 253 of chain B. Due to the preferred orientation of the complex molecules on the EM-grid, the resolution was limited to 4-5 Å at the interface between C3c and scFv, which still allows the replacement of the X-ray derived model into the EM map. Residues within 6 Å of the scFv were determined as binding site residues, corresponding to amino acids 366, 392-396, 413-421, 425, 427, 442, 453, 478 in human complement C3 (SEQ ID NO: 47). The results showed that clone I binds to an epitope that partially overlaps, but is not limited to, the compstatin binding site. The enhanced binding affinity to C3 exhibited by the molecules of the invention compared to compstatin derivatives is believed to result from the extension of this binding interface. These results indicate that the molecules of the invention bind to C3 in a different mode compared to prior art C3 inhibitors, which is believed to contribute to their unique ability to inhibit all three complement pathways, including the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP).

[0173] Example 10 In vitro BrM permeability assay and BrM permeability Bruch's membrane forms the innermost layer of the choroid, and structural changes are thought to cause complement activation in GA patients. Because complement-driven destruction of the choriocapillaris plays a major role in the pathophysiology of GA, excellent diffusion through Bruch's membrane is an important property of complement inhibitors for treating this disease. Therefore, we evaluated the ability of the scFv of the invention to diffuse through Bruch's membrane in vitro and compared it with Compound A2 and Compound A3. A. Materials and Methods Porcine Bruch's membrane samples are used for this measurement. Pig eyes were collected from the slaughterhouse within 6 hours after the animals were sacrificed and stored at 4° C. until use (same day).

[0174] Preparation of enriched Bruch's membrane (BrM): The eye is placed in a disposable Petri dish and any remaining tissue around the eyeball is removed. The eyeball is incised with a scalpel adjacent to the iris, and the front of the eye as well as the lens, vitreous and retinal tissue are removed from the eyecup. The enriched Bruch's membrane including the overlying RPE monolayer is gently removed from the entire inner surface of the eyecup and placed in a Petri dish filled with PBS and carefully flattened in the liquid. The Bruch's membrane is then transferred to an Ussing chamber, the Ussing chamber is closed and one compartment is filled with 1 ml PBS for leak testing. If no significant fluid accumulation is observed in the second compartment after 5 minutes, the membrane is considered intact. Bruch's preparations from 4 different eyes are used for each Ussing chamber. Incubation of enriched Bruch's membrane with test samples: Prepare a master mix of APL-2 surrogate (e.g., compound A2) and anti-C3 scFv or APL-2 surrogate (e.g., compound A2) and NGM621 surrogate (e.g., compound A3) in PBS (+0.02% NaN3 + protease inhibitor mixture (Complete Tablets, Mini EDTA-free, EASYpack; Roche)) for 4 Ussing chambers (1 ml per Ussing chamber) at a final concentration of 0.1 mg / ml for each compound. Add 1 ml of master mix per Ussing chamber to one compartment (referred to as the sample chamber). Add 1 ml of PBS (+0.02% NaN3 + protease inhibitor) per Ussing chamber to the second compartment (referred to as the diffusion chamber). Seal the Ussing chamber with parafilm and incubate at room temperature with gentle shaking to promote diffusion.

[0175] Sampling and membrane integrity analysis: For sampling at various time points (4 h, 24 h, 48 h and 72 h), the solutions in the single compartments were mixed by carefully pipetting up and down and 90 μl were transferred to collection tubes from each sample and diffusion chamber separately. At the end of the incubation period, membrane integrity tests are performed using Sphero Carboxyl Polystyrene Blue Particles: the suspension was diluted 1:10 in PBS and 200 ul per Ussing chamber was added to one compartment (referred to as sample chamber) and incubated for 30 min. The accumulation of blue particles in the second compartment is observed and camera images are recorded to control for overall membrane integrity. Sample analysis: 15 μl of each solution from the sample chamber and the diffusion chamber is effectively loaded onto two precast polyacrylamide gels (NuPAGE Bis-Tris gels) with a similar amount of stored master mix as a loading control. The first gel is analyzed by standard SDS-PAGE and Coomassie-based staining for protein detection (anti-C3 scFv). The second gel is analyzed by SDS-PAGE and barium iodide staining for PEG detection (compound A2).

[0176] Evaluation: The pictures of the SDS gels are analyzed using ImageJ2 software (Fiji). The bands are quantified and expressed as % of the test compound in each chamber, where 100% refers to the combined band intensity of the sample chamber and of the diffusion chamber of the same Ussing device. B. Results The ability to cross porcine BrM was compared for the scFv of the present invention and compstatin derivatives such as compound A2, which were incubated simultaneously on 4 different porcine eye BrM preparations.Compared to the APL-2 surrogate, a significantly higher amount of scFv crossed the BrM in all four membrane preparations.Similarly, the ability to cross porcine BrM was compared for the NGM621 surrogate (compound A3) and the APL-2 surrogate (compound A2), which were incubated simultaneously on 4 different porcine eye BrM preparations.

[0177] An exemplary antibody of the invention (clone I) demonstrated superior diffusion ability across Bruch's membrane compared to Compound A2 and Compound A3 (Figures 3 and 4). Compound A2: No penetration Compound A3: Reduced penetration compared to our exemplary antibody according to the invention ZIMURA®: Molecule not tested. Contains the same large 40 kDa PEG moiety as APL-2, so similar behavior is expected.

[0178] Example 11 Protein thermostability of the scFv of the present invention A. Materials and Methods Technology used: DSF Differential scanning fluorimetry (DSF) measures protein unfolding by monitoring changes in fluorescence as a function of temperature. Protein unfolding temperature (T m ) is related to antibody stability, especially aggregation during storage and long-term stability of therapeutic products. Thermal transitions of monoclonal antibody CH2 and CH3 domains are typically invariant for different antibodies within an isotype, with CH2 domain unfolding preceding CH3 domain unfolding.

[0179] B. Results The results are summarized in Table 21 below. [Table 21] High T m A high T value means that at a given temperature, a small fraction of the molecule exists in an unfolded state. m The high T value indicates that the enzyme maintains an active conformation at physiological temperatures. m The value is beneficial for therapeutic protein drugs. These results confirm the excellent pharmaceutical properties of the exemplary antibodies of the present invention. In particular, the improved thermal stability properties contribute to improved therapeutic efficacy while allowing for reduced injection doses and frequency to patients. In addition, the results show a long shelf life and improved stability in therapeutic products. The improved stability and prolonged activity contribute to making the antibodies of the present invention highly suitable for intravitreal administration.

[0180] Example 12 Immunogenicity risk: Screening for existing ADA We use bridging ELISA to screen the sera of healthy human donors for pre-existing ADA against clone I. Pre-existing ADA present in the sera from healthy human donors is bound by immobilized anti-C3 scFv. In the next step, ADA is bound by the same anti-C3 scFv labeled with biotin and detected by HRP-conjugated streptavidin.

[0181] A. Materials and Methods The scFvs are biotinylated using the "Biotin Conjugation Kit (Fast, Type A)-Lightning-Link" kit (Expedeon / Abcam) according to the supplier's instructions. Unlabeled scFvs are diluted to 0.5 μg / ml in PBS and 50 μl per well are transferred to a 96-well plate and incubated overnight at 4°C. The next day, the 96-well plate is washed with washing buffer (3×320 μl, 0.05% Tween 20 in PBS, pH 7.4) and nonspecific binding sites are blocked with 300 μl / well of blocking buffer (SuperBlock Blocking Buffer; Thermo Fisher Scientific) for 30 min at room temperature. The wells are then washed with washing buffer (3×320 μl / well).

[0182] Human serum from healthy individuals is diluted to 10% in PBS, 50 μl / well is added to a 96-well plate, and the plate is incubated for 1 hour at room temperature. The plate is then washed with wash buffer (3×320 μl / well) and 50 μl / well of biotinylated scFv (0.05 μg / ml in PBS) is added per well and incubated for 1 hour at room temperature. Plates are washed (3x320 μl / well with buffer) and 50 μl / well of Streptavidin PolyHRP40 (SDT Reagent; 1:5000 in PBS) is added and incubated for 30 minutes at room temperature. The plate is washed (3x 320 μl / well wash buffer) and 50 μl / well TMB substrate (Invitrogen) is added and incubated for 5 min at room temperature. The reaction is then stopped by adding 50 μl / well 1N H2SO4. Absorbance is measured at 450 nm. Pre-existing ADA results in an increase in absorbance. B. Results For clone I, pre-existing ADA was found in 9 of the 50 serum samples, indicating an overall low risk of immunogenicity. These results confirm that the antibodies of the present invention are highly suitable and promising for the treatment of ophthalmic or ocular diseases with only low immunogenicity risk.

[0183] Example 13 Biophysical characterization of the antibodies of the invention in highly concentrated formulations Storage stability Clone I, Clone II and Clone III are tested for storage stability. Samples are concentrated to 150 mg / ml (Vivaspin20 centrifugal device, 5 kDa MWCO at 3800×g) and stored at 25° C. and 40° C. for up to 4 weeks. Samples are analyzed for oligomerization (SE-HPLC) and chemical degradation (IEX-HPLC). The respective decrease in monomer content compared to the starting value is reported in Table 22. The starting monomer purity determined by SE-HPLC was 99.96% for each clone. The starting charge purity determined by IEX-HPLC was 98.07%, 97.16% and 96.76% for clones I, II and III, respectively. Clone I, Clone II and Clone III exhibited excellent monomericity and charge stability at 25° C. and 40° C. over a 4 week incubation period.

[0184] Viscosity at high concentrations The injection force required to deliver a drug through a standard intravitreal needle depends on the viscosity of the product and must be low enough to allow the physician to safely inject the product. Thus, a low viscosity, typically 15 mPa at 20° C., in a fully formulated product is required. * Less than a second is needed. Viscosity measurements are performed with samples prepared for storage stability studies. In addition to fully formulated samples (10 mM His pH 5.5, 275 mM sucrose, 0.02% w / v polysorbate 20), the viscosity of each candidate is also measured in minimal formulation (10 mM histidine pH 5.5). This is performed to evaluate the processability (e.g., filtration, pumping, diafiltration, etc.) of the candidate drug up to 125% of the final drug product concentration of 150 mg / ml.

[0185] Viscosity: 1000 seconds at 20°C -1 The shear rate is measured using a cone-plate rheometer at 1,000 s. -1 A stepwise increase in shear rate to 2000 s was performed with 100 data points collected. -1Further increases in shear rate to the starting rate are continued while collecting 10 more data points. Finally, a stepwise decrease in shear rate to the starting rate is performed. The resulting viscosity data is shown in Table 22. Good drug product handling properties in terms of filling and syringeability were observed for all candidates.

[0186] The viscosity and stability properties of clones I, II and III formulated at >150 mg / mL are summarized in Table 22. [Table 22] The inventors have demonstrated that exemplary antibodies of the invention exhibit exceptional stability in highly concentrated formulations.

Claims

1. An anti-C3 antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) and a variable light chain (VL), - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2 and 15, the CDR-H3 sequence of SEQ ID NO: 3; and - the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence selected from the group consisting of SEQ ID NO: 5 and 18, and the CDR-L3 sequence of SEQ ID NO: 6, Anti-C3 antibody or an antigen-binding fragment thereof.

2. - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 2 and the CDR-H3 sequence of SEQ ID NO: 3; and VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6, or - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 15 and the CDR-H3 sequence of SEQ ID NO: 3; and VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 5 and the CDR-L3 sequence of SEQ ID NO: 6, or - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 15 and the CDR-H3 sequence of SEQ ID NO: 3; and VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 18 and the CDR-L3 sequence of SEQ ID NO: 6, or - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence of SEQ ID NO: 2 and the CDR-H3 sequence of SEQ ID NO: 3; and VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence of SEQ ID NO: 18 and the CDR-L3 sequence of SEQ ID NO: 6, The anti-C3 antibody or antigen-binding fragment thereof according to claim 1.

3. - a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 20, 22 or 24; and a light chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 21, 23 or 25; The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 2,

4. - a heavy chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 20, 22 or 24; and a light chain variable region comprising an amino acid sequence at least 80%, at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of SEQ ID NO: 21, 23 or 25; Including, - VH comprises the CDR-H1 sequence of SEQ ID NO: 1, the CDR-H2 sequence selected from the group consisting of SEQ ID NO: 2 and 15, the CDR-H3 sequence of SEQ ID NO: 3; and - the VL comprises the CDR-L1 sequence of SEQ ID NO: 4, the CDR-L2 sequence selected from the group consisting of SEQ ID NO: 5 and 18, and the CDR-L3 sequence of SEQ ID NO: 6, The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 2.

5. - a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20, 22 or 24; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21, 23 or 25 The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 4,

6. a. a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO:20 and SEQ ID NO:21, respectively; b. a variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO:22 and SEQ ID NO:23, respectively; or c. A variable heavy chain and a variable light chain comprising the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:25, respectively. The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5,

7. The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antigen-binding fragment is selected from the group consisting of a single chain variable fragment (scFv), a Fab fragment, a Fab' fragment, an Fv fragment, a diabody and a small molecule antibody mimetic.

8. The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antigen-binding fragment is a single-chain variable fragment (scFv).

9. The anti-C3 antibody or antigen-binding fragment thereof of claim 8, wherein the antigen-binding fragment is a single-chain fragment comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 29, 30 and 31.

10. An anti-C3 antibody or an antigen-binding fragment thereof that binds to at least one amino acid residue selected from the group consisting of residues 366, 392-396, 413-421, 425, 427, 442, 453, and 478 of human complement C3 set forth in SEQ ID NO:

47.

11. An anti-C3 antibody or an antigen-binding fragment thereof that binds to all of amino acid residues 366, 392-396, 413-421, 425, 427, 442, 453, and 478 of human complement C3 set forth in SEQ ID NO:

47.

12. The anti-C3 antibody or antigen-binding fragment thereof according to claim 10 or 11, which is an anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

13. The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, which is capable of inhibiting complement activation pathways including the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP).

14. The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, which is capable of binding to complements C3 and C3b.

15. The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, which is capable of preventing the formation of C3 convertase.

16. The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, which is capable of penetrating Bruch's membrane.

17. Human C3, K D <50 nM, preferably K D <15 nM, preferably K D <10 nM, preferably K D < 7 nM, preferably K D <1 nM, preferably K D <0.5 nM, preferably K D <0.2 nM, preferably K D <0.15 nM, preferably K D <0.10 nM, preferably K D <0.05 nM, preferably K D <0.04 nM, or more preferably D The anti-C3 antibody or antigen-binding fragment thereof of any one of claims 1 to 16, which binds at <0.03 nM.

18. Human C3b, K D <50 nM, preferably K D <15 nM, preferably K D <10 nM, preferably K D < 7 nM, preferably K D <1 nM, preferably K D <0.5 nM, preferably K D <0.2 nM, preferably K D <0.15 nM, preferably K D <0.10 nM, or more preferably D The anti-C3 antibody or antigen-binding fragment thereof of any one of claims 1 to 17, which binds at <0.05 nM.

19. The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 18, having approximately equal binding affinity to C3 and C3b.

20. About 10 to C3a, iC3b, C4, C4b, C5 and / or C5b -4 The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, having a binding affinity of M or weaker than this.

21. The anti-C3 antibody or antigen-binding fragment thereof of any one of claims 1 to 19, having weaker binding affinity for C3a, iC3b, C4, C4b, C5 and / or C5b compared to its binding affinity for C3 and C3b.

22. The anti-C3 antibody or antigen-binding fragment thereof of any one of claims 1 to 19, which has no binding affinity for C3a, iC3b, C4, C4b, C5 and / or C5b.

23. An anti-C3 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 19, capable of inhibiting the C3 convertase amplification loop.

24. The anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, which is capable of inhibiting choroidal C3 activity.

25. The antibody or antigen-binding fragment according to any one of claims 1 to 24 for use as a medicament.

26. 25. The antibody or antigen-binding fragment of any one of claims 1 to 24 for use in the manufacture of a medicament.

27. 25. The antibody or antigen-binding fragment of any one of claims 1 to 24 for use in treating or preventing an ophthalmic or eye disease.

28. 25. The antibody or antigen-binding fragment of any one of claims 1 to 24 for use in the manufacture of a medicament for treating or preventing an ophthalmic or eye disease.

29. The disease may be retinopathy, proliferative retinopathy (PR) such as retinopathy of prematurity, diabetic retinopathy (DR) including ischemic retinopathy, proliferative diabetic retinopathy (PDR) and non-proliferative diabetic retinopathy, diabetic macular edema (DME), diabetic macular ischemia (DMI), age-related macular degeneration (AMD) including dry AMD and wet AMD, geographic atrophy (GA), retinitis pigmentosa, inherited retinal dystrophies, myopic degeneration, retinal vein occlusion, retinal artery occlusion, endophthalmitis, uveitis, cystoid macular edema, any retinal disease.

29. The antibody or antigen-binding fragment for use according to claim 27 or 28, wherein the antibody or antigen-binding fragment is selected from the group consisting of choroidal neovascular membrane secondary to glaucoma, optic neuropathy, glaucoma, retinal detachment, toxic retinopathy, radiation retinopathy, traumatic retinopathy, drug-induced retinal vasculopathy, retinal neovascularization, polypoidal choroidal vasculopathy, retinal vasculitis, retinal microaneurysms, retrolental fibroplasia, chorioretinitis, Fuchs' dystrophy, macular telangiectasia, Usher syndrome, paroxysmal nocturnal hemoglobinuria (PNH) and Stargardt's disease.

30. 30. The antibody or antigen-binding fragment for use according to any one of claims 27 to 29, wherein the ophthalmic or eye disease is selected from the group consisting of age-related macular degeneration, geographic atrophy, neovascular glaucoma and diabetic retinopathy.

31. The antibody or antigen-binding fragment for use according to any one of claims 27 to 30, wherein the ophthalmic or eye disorder is geographic atrophy.

32. 32. The antibody or antigen-binding fragment according to any one of claims 27 to 31 for use in treating an ophthalmic or eye disease by inhibiting the activity of the classical (CP), lectin (LP) and alternative (AP) pathways of complement, or by inhibiting the activity of choroidally localized complement C3.

33. A method for diagnosing a disorder associated with complement C3 in a biological sample using the anti-C3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 24.

34. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 24 and a pharma- ceutically acceptable carrier.

35. A pharmaceutical composition comprising one or more polynucleotides encoding the antibody or antigen-binding fragment of any one of claims 1 to 24 and a pharma- ceutically acceptable carrier.

36. The antibody or antigen-binding fragment thereof of any one of claims 1 to 24 or the pharmaceutical composition of claim 34, wherein the antibody or antigen-binding fragment thereof is administered by parenteral, intravenous, intravitreal or subcutaneous route of administration.

37. The antibody or antigen-binding fragment thereof of any one of claims 1 to 24 or the pharmaceutical composition of claim 34, wherein the antibody or antigen-binding fragment thereof is administered by the intravitreal route.

38. An isolated polynucleotide or polynucleotides encoding the antibody or antigen-binding fragment of any one of claims 1 to 24.

39. - a sequence encoding the heavy chain variable region according to SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: 24, and a sequence encoding the light chain variable region according to SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25 39. The isolated polynucleotide or plurality of polynucleotides of claim 38, comprising:

40. 40. An expression vector comprising the isolated polynucleotide or multiple polynucleotides of claim 38 or 39.

41. 41. A host cell comprising the isolated polynucleotide or polynucleotides of claim 38 or 39 or the expression vector of claim 40.

42. a. obtaining the host cell of claim 41; and b. Culturing the host cells A method for producing an anti-C3 antibody or an antigen-binding fragment thereof, comprising:

43. 43. The method of claim 42, further comprising recovering and purifying the antibody or antigen-binding fragment thereof.

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